Stability of casein micelles cross-linked with genipin: a physico-chemical study as a function of pH.

Casein micelles (CMs) are natural supramolecular aggregates present in milk. They are formed by the highly phosphorylated casein molecules, called αS1- αS2- β and ƙ- caseins [1]. Although CMs are quite stable against heat, their structure is highly sensitive to the ionic environment, especially acid pH. A possibility to stabilize CMs facing changes in the chemical environment is reticulation. Silva et al. [2] have stabilized CMs by using genipin (GP), a natural cross-linker extracted from Gardenia jasminoides [3]. The authors showed that the reaction was intramicellar, with formation of covalent bonds between GP and lysil - arginyl residues of CMs. In this context, the objective of the present work is to study the behavior of CMs cross-linked by GP as a function of pH under different stress conditions. Suspension of purified CMs (25 g∙L-1) was mixed with 10 mM of GP solution. Reaction between CMs and GP was carried out at 50°C for 24h followed by 4°C for 26h [2]. A control sample was prepared in the same conditions [2]. The samples were then aliquoted and acidified at different pH values from 7.0 to 2.0 with an interval of 0.5 pH unit at 4°C to prevent precipitation. After that, the temperature was slowly raised to 25°C and the samples were kept under stirring for 2 h. For each pH value, the charge ζ (Z-potential) and size of the particles were determined by dynamic light scattering. Heat coagulation time (HTC) at 140°C and ethanol stability (% ethanol v/v) were determined as a function of pH. CMs (control sample) precipitated below pH 5.5. CMs-GP precipitated from pH 3.5 to 4.5, whereas no precipitation was observed for the other pH values. Isoelectric point of CMs-GP was determined at pH 3.6, against 4.5 for CMs, by intersection of Z-potential curve on ordinate-axe. Highest stability was observed at pH 2, where visible coagulation of CMs-GP was observed only at 800 seconds at 140°C, against 40 seconds at pH 7 for CMs, or 87.5% (v/v) of ethanol against 50% (v/v) at pH 7 for CMs. We assume that GP increased stability of CMs against disruption by stabilization of polyelectrolyte brush of ƙ-casein. In order to confirm this hypothesis, we follow the action of chymosin on ƙ-casein of CMs and CMs-GP at neutral pH. Results showed gelification of CMs (control sample) at t=10 min, whereas no gelification was observed for CMs-GP after t=25 min of chymosin action. The potential use of CMs-GP as adaptable nanocarrier is proposed in this study.