Probing of Competitive Displacement Adsorption of Casein at Oil-in- Water Interface Using Equilibrium Force Distance Measurements V. Mahendran, J. Sangeetha, and John Philip* SMARTS, Metallurgy and Materials Group, Indira Gandhi Centre for Atomic Research, Kalpakkam 603 102, Tamil Nadu, India * S Supporting Information ABSTRACT: The equilibrium force distance measurement is employed for the first time to probe the competitive and displacement adsorption of casein at an oil-water (O/W) emulsion interface that was initially adsorbed with either a diblock polymer or an anionic surfactant. A significant change in the force-distance profile was observed under the competitive displacement adsorption of casein, which is further confirmed from the hydrodynamic diameter and zeta potential measurements. A decrease in the onset of repulsion and decay length are observed on competitive adsorption of smaller size casein molecules at O/W interface. With addition of casein in PVA-vac diblock polymer stabilized emulsion, the onset of repulsion decreases from 88 to 48 nm whereas the magnitude of force increases from 1 to 19 nN. The force decay length is reduced from 10.5 to 4.5 nm upon addition of casein. Our results suggest the complete replacement of adsorbed diblock polymers by casein molecules. The hydrodynamic diameter and zeta potential measurements corroborate the casein mediated polymer displacement and the competitive adsorption of casein at the O/W interface. In the case of anionic surfactant covered O/W interfaces, casein molecules weakly associate at the interface without displacing the smaller size surfactant molecules where no significant changes in the onset repulsion and force profiles are observed. These results suggest that the casein molecules are effective displacers for replacement of adsorbed macromolecules from formulations, which has several important practical applications. ■ INTRODUCTION Understanding the nature and reversibility of competitive displacement adsorption of smaller molecules at interfaces covered with macromolecules is important from practical applications point of view. 1 Such competitive displacement adsorption provides a unique possibility to reuse adsorbents. 2 As the competitive interactions between different adsorbing species determine the stability and product lifetime, several studies have been carried out on the interaction of protein with other molecules at the air-water and oil-water interface. 3-17 The recent neutron reflectivity studies show that a synergistic interaction between certain nonionic surfactants and proteins can lead to spontaneous self-assembly at the air-water interface to form layered surface structures. 14 Such protein-anionic surfactant interactions can also effectively used to tune electrostatic interactions. 15 The protein adsorption at the oil-water interface is important in manufacturing industries such as pharmaceutical and food emulsion industries. 14,18-20 Milk proteins are used as a stabilizer in food hydrocolloids because of their exceptional amphiphilic nature and stabilizing properties. 18,21 In bovine milk, casein consists of four major proteins, α s1 -casein, α s2 - casein, β-casein, and κ-casein, which are known to adsorb rapidly at the oil-water interface and provide stability via electrosteric stabilization. 22,23 α s1 -casein and β-casein are the major contents (∼75%) of milk casein. 22 The average molecular weight of β casein is ∼24 kDa with a net charge of -15e at neutral pH. β-Casein is an unstructured protein and has no internal covalent cross-links and has 209 amino acid residues, and the nonuniform distribution of hydrophobic and hydrophilic residues gives rise to a distinctly amphiphilic molecular structure. 24,25 β-Casein possesses a strong negative charge with four of the five phosphate groups being located at the N-terminal end between amino acid residues 13 and 21. 25 The average molecular weight of α s1 is ∼23.5 kDa with a net charge of -22e at neutral pH. 25,26 α s1 -Casein is also amphiphilic with nonuniform and more randomly distributed 199 hydrophobic and hydrophilic residues than that of β- casein. 24 β-Casein is more hydrophobic and surface active than α s1 -casein. 22 The casein resembles a block copolymer with alternating charge and hydrophobicity, that is a charged phosphopeptide loop and a hydrophobic train. 27 α s1 -Casein has an N-terminal hydrophobic train, followed by a charged loop and a final C-terminal hydrophobic train. 28 The k-casein Received: March 18, 2015 Revised: May 8, 2015 Article pubs.acs.org/JPCB © XXXX American Chemical Society A DOI: 10.1021/acs.jpcb.5b02612 J. Phys. Chem. B XXXX, XXX, XXX-XXX