How much can we trust polysorbates as food protein stabilizers - The case of bovine casein

Abstract

Polysorbates are widely used in food and cosmetic products and pharmaceuticals for emulsion stabilization, preventing surface absorption and as stabilizers against protein aggregation. But their impacts on the secondary structure and conformational stabilization of bovine casein, a typical intrinsically disordered protein (IDP), have not been fully addressed so far, which is crucial to evaluate the performance of polysorbates as stabilizer for food products containing bovine casein. Here, we assessed the effects of polysorbate 20 (PS-20) and polysorbate 80 (PS-80) on the secondary structure and conformation of bovine casein micelles by fluorescence and circular dichroism spectra, dynamic light scattering (DLS), small angle x-ray scattering (SAXS) and transition electron microscopy (TEM). In addition, three-dimensional structure of bovine β-casein, a major component of bovine casein, was predicted using homology modelling with the aid of PSIPRED server to outline the potential binding sites of bovine casein for polysorbates. We found that PS-20 presented a higher binding affinity (Ka = 3.76 × 106 M−1) and stoichiometry (n = 0.89) to bovine casein than that of PS-80 (Ka = 1.45 × 106 M−1, n = 0.45). The α-helical and β-sheet conformation of bovine casein was increased upon binding with PS-20 whereas binding of PS-80 had little impact on secondary structural elements of this milk protein. Moreover, bovine casein micelles were dissociated by PS-20, which co-assembled into micellar-like particles with a rather spherical shape and homogenous size distribution ranging from 20 to 50 nm in diameter. The lauric acid chains may bind with disordered regions connected by colloidal calcium phosphate (CCP) as the core, and the sorbitan groups may associate with N-terminal outwards as the shell providing hydrophilicity to stabilize the polysorbates-casein complex. On the contrary, PS-80 led to formation of sponge-like aggregates with the radius ranging from 200 to 500 nm as indicated by the data of DLS, SAXS and TME. We assume that the stearic hindrance originating from oleic group and the kink due to the cis-double bond in the sidechain of PS-80 contribute to the aggregation of bovine casein induced by PS-80. Furthermore, a tentative binding mechanism of polysorbates with bovine casein micelles and model of complexes were proposed to illustrate the polysorbates-induced structural change and conformational stabilization of bovine casein micelles. Thus, we argue that the difference in binding of polysorbates to bovine casein may influence the functional properties (emulsification, foaming ability, etc) of milk protein. Taken together, we suggest that PS-20 may be preferred option as potential protein stabilizer for dairy products, and careful consideration should be given when selecting PS-80 as stabilizer for bovine casein, a typical IDP.