Use of polysaccharides to control protein adsorption to the air–water interface
Introduction
The ability to create foams from aqueous protein solutions largely depends on the protein adsorption kinetics to the air–water interface. Adsorption kinetics of proteins have been extensively studied and large differences have been found between different proteins (e.g. β-lactoglobulin is known to quickly increase surface pressure (Paulsson & Dejmek, 1992), whereas for lysozyme the process is much slower (Tripp, Magda, & Andrade, 1995). Several steps in protein adsorption have been identified (Graham and Phillips, 1979, MacRitchie and Alexander, 1963a, MacRitchie and Alexander, 1963b): transport of the molecule to the interface by diffusion/convection, adsorption to the interface and possible conformational changes once adsorbed at the interface. The relation between adsorbed amount of protein at the interface, Γ (mg/m2) and the resulting surface pressure, Π (mN/m) is not linear. Below a certain minimum surface concentration, (depending on properties of the protein, e.g. net charge (Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2005), between 0.5 and 1 mg/m2) the surface pressure does not measurably deviate from zero and the surface pressure can be described by treating the adsorbed molecules as a two-dimensional ideal gas. When the surface concentration exceeds this typical minimum value, a steep increase in surface pressure is seen on further adsorption. Once Γ has increased until full monolayer coverage, the Π–Γ curve flattens (Benjamins, 2000). Obviously all this holds for pure protein solutions; but what happens when there are also polysaccharides, which are typically added to food systems to increase viscosity, present in the solution? This is likely to depend on the way the proteins and polysaccharides interact.
Protein/polysaccharide interaction is intensively investigated in a diversity of contexts: heparin and blood coagulation (Lee & Lee, 1995); protection of enzymes against high pressure or temperature; enzyme substrate binding and recovery and fractionation of milk proteins. A classical example from the food industry is the use of pectin to stabilize casein micelles in acidified milk drinks. Due to electrostatic interaction negatively charged pectin molecules adsorb at the casein micelles and prevent them from acid induced aggregation by electrostatic and steric repulsion (Maroziene and de Kruif, 2000, Pereyra et al., 1997, Syrbe et al., 1998). Also in food emulsions polysaccharides are used to prevent aggregation and creaming of emulsion droplets (Benichou et al., 2002, Dickinson, 2003). However, only little is known about the effect of protein/polysaccharide interactions on protein adsorption kinetics (Baeza et al., 2005, Dickinson and Pawlowsky, 1997) and this is only at pH above the iso-electric point of the protein. On mixing an anionic polysaccharide like pectin with a protein, four different regimes can be distinguished, depending on pH, ionic strength and mixing ratio, as described for mixtures of whey protein and arabic gum by Weinbreck, de Vries, Schrooyen, and de Kruif (2003). At neutral pH and low ionic strength (less than 50 mM) both the protein and the polysaccharide are negatively charged and although there can be some attractive interaction between the positively charged groups on the protein and the negatively charged polysaccharide, the components are cosoluble (I). On decreasing the pH close to the iso-electric pH of the protein or below, soluble protein/polysaccharide complexes are formed (II). Further decrease of the pH leads to aggregation of the soluble complexes and subsequently complex coacervation (III). At pH values below 2.5 complexation can be suppressed by protonation of the acidic groups on the polysaccharide (IV) (Weinbreck et al., 2003). From the work of Girard, Turgeon, and Gauthier (2003) we know that also β-lactoglobulin and pectin can form soluble complexes around pH 4.5. Protein adsorption kinetics to the air–water interface from mixed protein/polysaccharide solutions depend on the extent of protein complexation to pectin in the bulk, which in turn depends on parameters like pH, ionic strength, mixing ratio and charge density of the ingredients. This paper aims at understanding how one can control protein adsorption kinetics (as monitored by drop tensiometry) and, with that, foam formation by manipulating protein/polysaccharide interaction in the bulk.
Section snippets
Materials
Acetate buffer and NaCl solutions were prepared from analytical grade chemicals and deionised water. Bovine β-lactoglobulin was purified using a non-denaturing method as described previously (de Jongh, Gröneveld, & de Groot, 2001) Ovalbumin was isolated as described before (Wierenga, Meinders, Egmond, Voragen, & de Jongh, 2003) Lysozyme L-6878 was purchased from Sigma-Aldrich (St Louis, Missouri, USA) and used without further purification. Stock solutions of 0.2 mg/ml protein were prepared by
Results
To monitor the adsorption kinetics of protein in presence and absence of polysaccharides, surface pressure at the air–water interface was measured as a function of time for β-lactoglobulin (β-Lg) solutions and mixed β-lactoglobulin/pectin solutions, at various pH values (Fig. 1). The protein concentration of all samples was 0.1 g/L, protein/polysaccharide mixing ratio was kept constant at 2 w/w and ionic strength was kept low (on the order of 1 mM, samples were matched on the basis of a
Discussion
Since, in the pH region (II) above the pK of a polysaccharide and below the iso-electric point of the protein both components are oppositely charged, they electrostatically attract each other. From literature it is known that this can lead to the formation of soluble complexes. (Weinbreck et al., 2003). Complexation of protein molecules to pectin reduces the internal repulsion of the pectin molecule leading to a smaller hydrodynamic radius and an increase in the intensity of scattered light as
Acknowledgements
We acknowledge Bram Sperber from the Laboratory of Food Chemistry, Wageningen University, the Netherlands for his assistance with dynamic light scattering measurements.
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