New insights about flocculation process in sodium caseinate-stabilized emulsions

Highlights

• Emulsions with the same formulation destabilized by different mechanisms

• Flocculation depended on casein molecules aggregation in different environments.

• SAXS data were fitted by a three region model that considered polydispersity.

• Structural parameters were calculated from the model.

• Structural changes in casein entities were related to macroscopic properties.

Abstract

Flocculation process was studied in emulsions formulated with 10 wt.% sunflower oil, 2, 5 or 7.5 wt.% NaCas, and with or without addition of sucrose (0, 5, 10, 15, 20 or 30 wt.%). Two different processing conditions were used to prepare emulsions: ultraturrax homogenization or further homogenization by ultrasound. Emulsions with droplets with diameters above (coarse) or below (fine) 1 μm were obtained. Emulsions were analyzed for droplet size distribution by static light scattering (SLS), stability by Turbiscan, and structure by confocal laser scanning microscopy (CLSM) and small angle X-ray scattering (SAXS). SAXS data were fitted by a theoretical model that considered a system composed of poly dispersed spheres with repulsive interaction and presence of aggregates. Flocculation behavior was caused by the self-assembly properties of NaCas, but the process was more closely related to interfacial protein content than micelles concentration in the aqueous phase. The results indicated that casein aggregation was strongly affected by disaccharide addition, hydrophobic interaction of the emulsion droplets, and interactions among interfacial protein molecules. The structural changes detected in the protein micelles in different environments allowed understanding the macroscopic physical behavior observed in concentrated NaCas emulsions.

Introduction

Caseins are a family of unstructured amphiphilic milk proteins known as α_S1, α_S2, β and κ, which have the tendency to naturally form complex aggregates with reported average sizes of up to 300 nm (Livney, 2010). Sodium caseinate (NaCas) results from the removal of calcium phosphate and is widely used in pharmaceutical and food industries as an emulsion stabilizer and foam formation agent. NaCas exists in aqueous solution at neutral pH as a soluble mixture of casein monomers and self-assembled protein aggregates of sizes around 10–20 nm in diameter and molecular weight of ~ 2.5 × 10⁵ Da, also called nanoparticles in literature (Dickinson, 2006).

Among other factors, the extent of aggregation of NaCas is affected by the presence in the aqueous phase of compounds such as disaccharides. The effect of sucrose on emulsion physical behavior was reported to be strongly dependent on the pH. At neutral pH light scattering studies indicated a reduction in aggregation but at pH close to the isoelectric point (Ip = 4.5) an increase on the radius of gyration was reported (Belyakova et al., 2003, Dickinson, 2006, Ruis et al., 2007). These results indicated that sucrose has an important effect on this system and therefore it is also of great interest to investigate how disaccharides modify the structure of sodium caseinate stabilized emulsions.

In order to obtain unbiased results when studying emulsion stability as well as structural properties, the experimental techniques applied have to be as little invasive as possible. Scattering methods are a good example of such techniques and can provide important information on the structural and dynamical properties of heterogeneous fluids (Alexander & Dalgleish, 2006). However, some of those techniques require a substantial dilution of the samples. This dilution disrupts emulsion structures modifying the actual system. Therefore, the ability to study the stability of food emulsions in their undiluted forms may reveal subtle correlations affecting emulsions physical changes. A relatively recently developed technique, the Turbiscan method, allows registration of the turbidity profile of an emulsion along the height of a glass tube filled with the emulsion. The analysis of the turbidity profiles with time leads to quantitative data on the stability of the studied emulsions and allows making objective comparisons between different emulsions (Mengual, Meunier, Cayre, Puech, & Snabre, 1999a). Food emulsions that can be described by the Turbiscan method have droplet sizes typically between 0.1 and 100 μm (Huck-Iriart et al., 2011, Huck-Iriart et al., 2013). Small Angle X-Ray Scattering (SAXS) is a non-invasive technique generally used in materials and life science. Due to the short wavelength of X-Rays, this technique provides structural information on colloidal particles and complex fluids in the 1–100 nm size range. Combining both techniques, Turbiscan and SAXS, experimental data may be obtained in a wide range of sizes.

For the correct analysis and interpretation of the SAXS data, a possible approach is to assume a certain model for the system, based on previous knowledge, and compare it with the measured data using least square methods (Pedersen, 1997). In the past, simple models were used to describe milk casein in dilute solution (Holt, Kruif, Tuinier, & Timmis, 2003) and some important structural information was reported. However, in emulsions system, the detailed nature of the molecular interactions involved in the casein micelles formation and the arrangement of the proteins in the casein micellar aggregates is still not completely established. Notwithstanding, the existing data serves as a basis to propose a working micellar model that describes the structure of more complex systems, such as NaCas oil-in-water (O/W) emulsions.

Emulsions are thermodynamically unstable liquid-liquid dispersions. The common strategies to kinetically stabilize these systems involve modification of the liquid-liquid surface tension, the droplet size and the viscosity of the continuum phase (Ivanov & Kralchevsky, 1997). These strategies are not independent since a complex interaction between proteins, surfactants, additives and liquid phases might occur (Jourdain et al., 2009, Woodward et al., 2009). Stability of sodium caseinate emulsions has been widely investigated (Dickinson et al., 1997, Hemar et al., 2003, Belyakova et al., 2003, McClements, 2004), to name a few. Flocculation and creaming were the most frequent macroscopic manifestations reported. According to the literature, flocculation occurred above some critical concentration of stabilizing polymer and was sensitive to its concentration. Flocculation was especially related to the excess of unadsorbed protein (Dickinson et al., 1997, Dickinson and Golding, 1997). Although flocculation mechanism was widely investigated, recent studies using non disturbing techniques showed that flocculation process was more complex than reported in literature (Álvarez-Cerimedo et al., 2010, Huck-Iriart et al., 2013). The results reported in those studies suggest that in sodium caseinate-stabilized emulsions, flocculation is a process that remains fairly poorly understood.

The aim of the present work was to further study flocculation process at the micro and nanoscales from a few nanometers up to hundreds of microns.