Influence of storage condition and sample age on the rennet induced aggregation of para-casein micelles

The growing use of novel casein-based functional materials beyond traditional food applications requires a detailed understanding of how storage conditions affect the aggregation behaviour of casein micelles. In this study, we systematically varied storage time (2, 7, 12 and 79 days) and temperature (5, 10 and 15 °C) as well as mechanical treatment (stirred vs. unstirred) and then monitored enzymatically induced aggregation by means of turbidity measurements. The aggregation kinetics were analysed using an asymmetric sigmoidal fitting model, which showed that the delay time and the time to reach the maximum aggregation rate generally decreased with increasing storage time and temperature. The maximum aggregation rate increased up to a storage period of 12 days, but longer storage – especially at elevated temperatures – resulted in reduced rates, suggesting structural rearrangements, pre-aggregation or partial micelle degradation. Stirring during storage had a temperature-dependent and time-dependent effect. At moderate temperatures and medium storage times, it generally resulted in a more homogeneous micelle dispersion and prevented reversible pre-aggregation, while at low temperatures and very short storage times, it helped to keep aggregation rates lower and samples more stable than in non-stirred controls. At elevated temperatures and longer storage times, stirring promoted irreversible aggregation prior to enzyme addition. Differential dynamic microscopy measurements of hydrodynamic radii prior to enzyme addition confirmed the presence of pre-aggregates that could be broken up by stirring, as well as irreversible aggregates that formed during prolonged storage at higher temperatures.