β-Lactoglobulin and WPI aggregates: Formation, structure and applications

Abstract

The literature is reviewed on the formation and the structure of β-lactoglobulin and whey protein isolate (WPI) aggregates in aqueous solution induced by heating. The focus is on the effects of the pH and added salt. The use of β-lactoglobulin and WPI aggregates in cold-set gels, foams and emulsions, encapsulation, and films and coatings is also reviewed.

Introduction

Globular proteins have a dense well-defined and mainly rigid structure that determines its function in living organisms. In aqueous solutions globular proteins are usually present in the form of monomers or small oligomers and they are stabilized by electrostatic repulsion. The net charge of globular proteins depends on the pH and is zero at the iso-electric point (pI). However, often the proteins contain both negative and positive charges. Heating or pressurizing may cause a change of the structure and make the peptide chain more mobile. As a consequence, segments of different molecules may interact through hydrophobic interactions or by forming hydrogen bonds, leading to aggregation. If the proteins contain cysteine, intermolecular disulfide bridges may be formed.

Often heat or pressure induced aggregation of proteins is found to be irreversible and the aggregates persist at room temperature and atmospheric pressure. The size and the structure of the aggregates depend on the protein concentration (C), the heating protocol, the type and concentration of added salt, and the pH. The system may also gel above a critical concentration (C_g) that depends on the type of protein and the external conditions. In general, C_g is lower when electrostatic repulsion between the proteins is weaker.

Here we review the formation, structure, properties and applications of aggregates formed by β-lactoglobulin (β-lg) and by whey protein isolate (WPI). β-lg, which represents about 60% of the proteins in bovine whey, is a globular protein with a radius of about 2 nm, a molar mass of 18.2 kg mol⁻¹. It contains two disulfide bridges and one free thiol (Hambling, Mc Alpine, & Sawyer, 1992). In milk mainly two genetic variants A and B are found in approximately equal quantities. Most investigations have been done on natural mixtures of both variants. Studies of pure variants have shown subtle differences especially concerning the rate of aggregation (Croguennec, O’Kennedy, et al., 2004, Le Bon et al., 2002, Manderson et al., 1999), but they will not be discussed here. α-lactalbumin (α-la) is the second most prevalent protein in bovine whey (20%) and is a globular protein with a molar mass of 14.2 kg mol⁻¹. It contains four disulfide bonds, but no free thiol group.

Long range electrostatic repulsion renders native β-lg solutions stable except close to the iso-electric point (pI ≈ 5.1) where aggregation at room temperatures has been observed (Majhi et al., 2006, Mehalebi et al., 2008b, Schmitt et al., 2009, Stading and Hermannsson, 1990). The extent and the rate of aggregation were shown to strongly decrease when NaCl was added (Majhi et al., 2006). Close to pI the proteins contain charges with both positive and negative sign and interaction between charges with opposite sign may be involved in the aggregation besides hydrophobic interaction and hydrogen bonds. The rate of aggregation is very slow at room temperature and may be imperceptible at low protein concentrations. However, it increases with increasing temperature and can lead to precipitation of a significant fraction of the proteins at high concentrations (Mehalebi et al., 2008b). Aggregation of native β-lg is completely reversed when the pH is increased or decreased away from pI. The rate of aggregation is maximum at about pH 4.6 (Majhi et al., 2006) and is negligibly slow for pH 5.5 and higher even at 50 °C and C = 100 g L⁻¹ (Mehalebi et al., 2008b).

Aggregation of native β-lg is favoured by adding salt. Unterhaslberger, Schmitt, Sanchez, Appolonia-Nouzille and Raemy (2006) observed in DSC experiments that at high salt concentrations an exothermic peak corresponding to protein aggregation shifted to lower temperatures than the well-known endothermic peak corresponding to β-lg unfolding. The effect was more pronounced at pH 4.0 than at pH 7.0.

Except in a small pH range around pI or at high ionic strength, native β-lg is present as monomers and dimers in equilibrium. The equilibrium is shifted towards the monomer with increasing temperature, increasing charge density and decreasing ionic strength (Apenten and Galani, 2000, Aymard, Durand, et al., 1996, Renard et al., 1998, Verheul et al., 1999). When β-lg is heated above about 60 °C its secondary structure is partially modified (Cairoli et al., 1994, Carrotta et al., 2001, Croguennec, Molle, et al., 2004, Iametti et al., 1995, Iametti et al., 1996, Manderson et al., 1998, Manderson et al., 1999, Ochenduszko and Buckin, 2010, Qi et al., 1997). This state is often referred to as molten globule, because the protein chain is more mobile, but does not unfold completely. Groups that in the native state are buried inside become exposed and may interact with other proteins. As a consequence, bonds are formed between proteins leading to aggregation. The rate at which native β-lg aggregates increases exponentially with increasing temperature and is characterized by a large activation energy (Le Bon, Nicolai, & Durand, 1999b). It varies from imperceptibly slow to almost instantaneous over a temperature range of about 30 °C.

The size of the aggregates increases with heating time until steady state is reached and no native protein is left (Le Bon et al., 1999a, Le Bon et al., 1999b). The steady state is usually a stable dispersion of aggregates or a gel, but in some situations the gels are macroscopically heterogeneous or the aggregates precipitate. The characteristics of the aggregates strongly depend on the pH and the presence of added salt.

In the following, we will first discuss the influence of the pH and the ionic strength on the kinetics of the aggregation. We will compare aggregation in pure β-lg solutions with that in binary mixtures with α-la and in WPI. Then, we will discuss the structure of the protein aggregates obtained. The last section will review the most relevant food applications of β-lg and WPI aggregates.