Crosslinking of casein micelles by Genipin

Casein micelles (CM) are porous colloidal particles of about 150 nm of diameter, composed of different casein molecules and calcium phosphate. Their modifications, by technological treatments and/or addition of bio-chemical reagents, constitute a scientific challenge to create new structures and functionalities. In this work, modification of CM by a natural cross-linker, named genipin, was performed and their new physico-chemical properties were appreciated. Genipin (5, 10 and 20 mM) were added to micellar suspensions of casein at 25 g.l-1, previously dispersed in a buffer system consisting of 25 mM HEPES and 2 mM CaCl2 at pH 7.15. The reaction was carried out at 50 °C for 24 h. A control sample was treated in the same conditions. The experiment was done 3 times independently. The genipin reaction was monitored by changes in UV/visible-spectra between 190-900 nm during incubation at 50 °C. After that, the reacted products were evaluated by reversed-phase high pressure liquid chromatography (RP-HPLC) and sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS-PAGE). Chromatography and electrophorese were performed under dissociation conditions. The reaction level was estimated by the measurement of the available lysine and arginine. The consequences of the cross-linking on the colloidal properties were investigated by measuring: zeta potential and size distribution by dynamic light scattering; hydration after pellet formation by ultracentrifugation; shape by scanning electron microscopy (SEM); viscosity at shear rates lower than 100 s-1; and surface tension by pendant drop method. The reaction between genipin and primary amines was characterized by formation of blue pigmented products, presenting a maximum absorption at ~600 nm. Higher were the genipin concentrations, more intense was the light absorption. The RP-HPLC profiles showed that above 5 mM of genipin, it was not possible to observe individual casein molecules. SDS-PAGE gels confirmed these results and revealed that casein molecules formed polymers with molecular weights greater than 200 kDa. The analysis of amino-acids highlighted that lysine was mainly involved in cross-linking, with a minor participation of arginine. By comparing to control sample, the percentage reduction in the concentrations of lysine and arginine reached 95 and 12 %, respectively, for 20 mM of genipin. The zeta potential and hydration values of CM were gradually reduced, indicating changes in their surface properties. The size distribution and SEM did not revealed major changes regarding the average diameter and shape of the CM. Internal crosslinking was confirmed by submitting the CM to dissociation conditions, followed by ultracentrifugation and analyses of the mass of pellet obtained. Regarding the viscosity, all the samples behaved as Newtonian fluids, nevertheless the values decreased progressively according to cross-linking intensity. Contrarily, the final values of surface tension as well as the adsorption rates at air/water interfaces increased gradually. Thanks to this new reagent, it was possible to create originally modified CM. Further researches are necessary to elucidate the modifications in their internal structure and the consequences in their functional properties, like gels, emulsions and foams