Protein adsorption at oil-water interfaces interfacial structure and function of proteins in emulsions
2017-05-18T02:51:33Z (GMT) by
Protein adsorption at oil-water interfaces of emulsions is an important process in the manufacture of many emulsion-based products in food and pharmaceutical industries. It is widely known that for protein-stabilized oil-in-water emulsions, the interfacial structure of proteins adsorbed at oil-water interfaces plays an active role in controlling the physical stability of the emulsions. However, the colloidal properties of emulsions such as the strong scattering and high absorbance of light have severely limited the ability of spectroscopic techniques to measure the interfacial structure of proteins. The lack of studies that systematically investigate the interfacial structure of proteins adsorbed at oil-water interfaces has limited our understanding of how the adsorption process mediates protein structural change and in turn the physical stability of the protein-stabilized emulsions. To address this issue, there is therefore great need to employ novel biophysical techniques that provide molecular level understanding of the interfacial structure of proteins adsorbed at oil-water interfaces. This thesis explores three biophysical techniques, i.e. synchrotron radiation circular dichroism (SRCD) spectroscopy, front-face tryptophan fluorescence (FFTF) spectroscopy and dual polarization interferometry (DPI), to study the interfacial structure of proteins adsorbed at oil-water interfaces of emulsions. The strong light from the synchrotron radiation source significantly enhanced the sensitivity of SRCD spectroscopy in measuring optically turbid emulsions. SRCD spectroscopy proved to be a powerful technique providing information on both the secondary and tertiary structure of proteins adsorbed at oil-water interfaces. Studies using FFTF spectroscopy in this thesis provided specific information on changes in the tertiary folding of globular proteins adsorbed at oil-water interfaces. DPI was employed in this thesis as a sensing technique to measure the geometric dimensions of proteins (thickness and density) adsorbed at planar hydrophobic interfaces mimicking oil-water interfaces of emulsions. Using the techniques of SRCD, FFTF and DPI, the interfacial structures of three proteins, i.e. β-lactoglobulin (β-Lg), α-lactalbumin (α-La) and β-casein, adsorbed at oil-water interfaces were therefore characterized at a molecular level. These milk proteins are common components of emulsifiers used in many food emulsions and have been frequently used as model proteins in studies of protein adsorption. Studies on the interfacial structure of β-Lg adsorbed at oil-water interfaces of two model emulsions (hexadecane-in-water and tricaprylin-in-water emulsions) showed that upon adsorption to oil-water interfaces, β-Lg changed from its native globular structure (3.6 nm in diameter) rich in β-sheet into an open flat structure with a non-native secondary structure with heat-resistant, α-helical-rich characteristics. The β-Lg layer adsorbed at planar hydrophobic surfaces mimicking oil-water interfaces of emulsions was shown to be thin (~1 nm) and dense (~1 g/cm3). The droplet surfaces surrounded by the β-Lg interfacial layers were negatively charged (approximately -60 mV of ζ-potential), providing electrostatic repulsion between the emulsion droplets. Parallel studies on the physical stability of β-Lg-stabilized emulsions showed that these emulsions were resistant to droplet flocculation under the condition of heating up to 90 °C. However, increasing the ionic strength by adding 120 mM NaCl reduced the strength of the electrostatic interactions and thus caused β-Lg-stabilized emulsions to undergo heat-induced droplet flocculation. Studies on the interfacial structure of α-La adsorbed at oil-water interfaces showed that α-La also underwent a significant structural change. Adsorption caused a loss of the well-defined globular structure of α-La (2.5 x 3.2 x 3.7 nm in crystal structure dimension) accompanied by the formation of a non-native, heat-resistant, α-helical-rich secondary structure. This structural change resulted in a thin (~1 nm) and dense (~1 g/cm3) layer of α-La, which was negatively charged (approximately -49 mV of ζ-potential). The main stabilization mechanism of α-La-stabilized emulsions was therefore also electrostatic repulsion. Like β-Lg-stabilized emulsions, α-La-stabilized emulsions were resistant to droplet flocculation under the condition of heating up to 90 °C. Interestingly, α-La-stabilized emulsions displayed better stability than β-Lg-stabilized emulsions under the condition of heating in the presence of 120 mM NaCl, mainly due to the lack of inter-droplet disulfide bonding reactions between the interfacial α-La layers. Studies on β-casein, a flexible protein with a large amount of unordered secondary structure, showed that adsorption to oil-water interfaces induced the formation of the non-native α-helical structure in β-casein. The β-casein layer adsorbed at the planar hydrophobic surface was thicker (~5 nm) and more diffuse (0.4 g/cm3) than the β-Lg and α-La layers. The droplet surfaces surrounded by the β-casein layers were also negatively charged (-40 mV of ζ-potential). This interfacial structure of β-casein provided both electrostatic and steric repulsion between the droplets of β-casein-stabilized emulsions, which showed excellent resistance to droplet flocculation under conditions of heating up to 90 °C and 120 mM NaCl addition. This interfacial structure and function of β-casein in oil-in-water emulsions led to a study exploring the ability of β-casein as a stabilizer in another colloidal system, i.e. lipid liquid crystalline nanostructured particles, which are important systems in drug delivery applications. Results obtained from this study revealed that β-casein adsorbed to lipid-water interfaces, provided steric stabilization and favoured the formation of the hexagonal liquid crystalline phase in these lipid nanostructured particles. Overall, studies in this thesis have advanced our understanding of how adsorption to oil-water or lipid-water interfaces leads to changes in the structure of proteins and in turn how it impacts on their function as stabilizers for colloids containing oil or lipid particles, which have implications in areas such as improvement of food emulsion stability and development of new drug delivery systems.