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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.

Avaliaram-se os métodos de eletroforese em gel de poliacrilamida (PAGE) em presença de uréia (uréia-PAGE) e dodecil sulfato de sódio (SDS-PAGE) para identificar a adulteração do leite de cabra pela adição do leite de vaca. Um método foi otimizado para preparação do caseinato de sódio em poucos minutos para análise eletroforética. Uréia-PAGE foi o método mais apropriado para identificação desse tipo de fraude, em decorrência da presença da caseína alfas1 com migração mais rápida no leite bovino. A presença da alfas1-caseína bovina foi detectada a partir da adição de 2,5% de leite de vaca utilizando uréia-PAGE. O limite de detecção, a repetibilidade, o tempo para execução indicaram que esse método pode ser aplicado como rotina no controle de qualidade do leite de cabra recebido pelas indústrias de processamento. Polyacrylamide gel electrophoresis (PAGE) in presence of urea (urea-PAGE) or sodium dodecyl sulfate (SDS-PAGE) was evaluated to detect the presence of cow milk added to goat milk. A method was optimized to prepare sodium caseinate from milk in few minutes. After that, the sodium caseinate was analyzed by PAGE. The urea-PAGE was the most appropriated method to identify adulteration as caprine and bovine alphas1-caseins displayed different migration rates. When cow milk was added to goat milk at different proportions, the presence of bovine alphas1-casein was detected in the mixture by urea-PAGE for a minimal proportion of 2.5% of cow milk added to goat milk. The good sensitivity, the repeatability and the short time for execution indicate that the described method will be able to be routinely applied for the quality control of goat milk in dairy industry.

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International audience

International audience

International audience