Foaming and emulsifying

WP are efficient foaming and emulsifying agents. The amount of sulfhydryl groups, molecular flexibility, hydrophobicity and surface activity determine their ability to form stable emulsions and foams (Lee et al., 1992). The optimum foaming and emulsifying characteristics of the proteins depend on how efficiently they diffuse to the newly formed interface, get unfolded and reorient to lower interfacial tension and form cohesive and viscoelastic films by polymerisation mainly via disulfide bonds and hydrophobic interactions (Monahan et al., 1993; Lee et al., 1992; Bouaouina et al., 2006). By increasing the ratio of β-Lg to α–La,

the foam yield stress of whey protein products can be modified (Foegeding et al., 2002). Sulfhydryl groups, which are more active at pH 7, contribute to molecular flexibility while disulfide bridges contribute to rigidity. Molecular flexibility enhances emulsion and foam formation by increasing the rate of unfolding at the interface and allowing more favourable alignment of polar and non polar groups in their preferred phase (Klemaszewski and Kinsella, 1991). At pH 7, whey proteins have a net negative charge, but at pH 8 possibly due to oxidation of thiol group, they easily form disulfide bonds. Proteins at an appropriate pH with increased net negative charge repulse each other creating a barrier to the close approach of droplets, thereby retarding the rate of coalescence and resulting in more stable emulsions (Klemaszewski and Kinsella, 1991). Previous studies have revealed that there is an improved overrun and increased stability of foams at pH 7 when heated to 55ºC (Phillips et al., 1990). At higher temperatures, however, foaming and emulsifying characteristics may be impaired due to protein aggregation, which decreases the availability of proteins to form films and emulsions (Phillips et al., 1990). Furthermore, since WP are even soluble at their isoelectric points (pH ~ 5), they exhibit superior foaming properties having better protein-protein interactions to form stable foams around that pH. In addition, the

β-Lg B variant appeared to adsorb more rapidly at interfaces than variant A although β-Lg A and B variants vary by only two amino acids (Foegeding et al., 2002).

2.4.4 Gelation

A gel is an intermediate state between a solid and a liquid which is also defined as ‘a substantially diluted system that exhibits no steady state flow’ (Damodaran et al., 2008). The capability of undenatured WP to form stable heat induced gels is also very important in food formulations and product development as these gels can act as a basic medium to hold other components such as water, lipids, sugars, flavour in heterogeneous food systems providing textual and thickening attributes

(Matsudomi et al., 1993; Resch et al., 2004). Compared to pre-gelatinized starches and hydrocolloids, which also have the capacity of water holding and increasing viscosity, whey proteins have added superior nutritional benefits (Resch et al., 2005). Thermal gelation involves initial unfolding and subsequent aggregation of proteins depending on the balance of attractive and repulsive forces (Hudson et al., 2000). The irreversible unfolding of whey proteins occur at temperatures > 60ºC (Considine et al., 2007). The process involves a series of physicochemical reactions such as dissociation, denaturation and exposure of hydrophobic residues and resulting polymerization via formation of intermolecular disulfide bonds, hydrophobic, hydrogen and ionic bonds (Brandenberg et al., 1992).

A proper balance of protein-protein as well as protein-water interactions fundamentally controls gelation mechanism and the gel appearance. If the protein- protein interactions are much greater than the protein-water interactions mostly a precipitate is formed. On the contrary, if protein-solvent interactions predominate the system would not gel. Another vital factor for the formation of a self-stranded gel network is the protein concentration. The least concentration end point (LCE) is the minimum protein concentration required to form such a network at given conditions (Gosal and Ross-Murphy, 2000; Damodaran et al., 2008). The physical characteristics of these gels can be altered by manipulating protein concentration, ionic strength, heating time and temperature, solvent condition and quality and addition of other macromolecules or filler particles (Verheul et al., 1998).

When there are large electrostatic repulsions between the proteins, i.e., at low ionic strength and far from the isoelectric point (pI) of the proteins, transparent, fine stranded gel structures are formed. At pH > pI fine stranded, strong elastic gels are formed and at pH < pI fine stranded weak brittle gels are formed. At high ionic strength and around isoelectric point of the proteins, turbid, milk white and particulate gel structures are formed (Verheul et al., 1998).

Figure 2.2 Physicochemical changes that take place during formation of viscoelastic gel network during heating of native whey proteins with simultaneous occurrence of steps (i), (ii) and (iii) (Based on Alting, 2003).

Dissociation

Unfolding

Gel network

of proteins

and solvent

Aggregation

Gelation

Native

whey

proteins

Figure 2.3 Model of globular protein gelation depicting changes from native protein to slightly unfolded (.) and more extensively unfolded (..) states, and resultant gel types. (Adapted from Foegeding, E. A., (2005), Rheology, Structure and Texture Perception in Food Protein Gels in Food colloids: Interactions, Microstructure and Processing).

In addition, the optimum pH for gel formation is about 7-8 for most proteins (Dickinson, 2005; Verheul et al., 1998; Damodaran et al., 2008). The translucent gels, formed primarily by hydrogen bonding, hold more water than coagulum or particulate gels and show less syneresis. In these gels, water is hydrogen bonded to carbonyl and N-H groups of the peptide bonds and water may exist as a hydrogen bonding cross linker between C=O and N-H groups of peptide segments probably restricting the flowability of water (Damodaran et al., 2008). Furthermore, a number of hydrophobic residues in the protein also determine the type of forming gel. The general trend is that the proteins with more than 31.5 mole percentage of hydrophobic residues form coagulum type gels while those with less than 31.5 mol%, usually form translucent gels in the aqueous medium (Damodaran et al., 2008).

The cold-set whey protein gelation is also useful as a novel and alternative method in formulating food products with modified functionalities (Marangoni et al., 2000). It is mostly important in preparations of heat-sensitive food products with delicate texture and flavour (Thompson et al., 2009). In comparison with heat-induced gelation, where the processes of denaturation, aggregation and gelation steps simultaneously occur, the cold gelation occurs in two separate steps as denaturation (i) and activation of proteins and gelation (ii) (Alting, 2003) as shown bellow.

Native

whey

proteins

Denaturation

& activation

Whey

protein gel

network

Usually, the activation of protein molecules is achieved by heat-denaturation, at a protein concentration below the critical gelation concentration, and at a low ionic strength and/or far from the isoelectric point (normally at neutral pH). Consequently proteins undergo structural rearrangements, including unfolding and conversion into small soluble aggregates. Also, upon cooling, they remain soluble without gelling. Strong electrostatic repulsions between proteins and negligible salting-out effects prevent gelation of the proteins. The gelation is then accomplished at cold-set conditions by changing the solvent quality with addition of salts or adjustment of pH to screen repulsive forces or by bringing the pH towards the isoelectric point, respectively (Thompson et al, 2009; Alting et al., 2000; Marangoni et al., 2000). However, the direct addition of salt or acid does not yield a viscoelastic protein network.

The salts such as sodium chloride or calcium chloride can be added to enhance protein aggregation via screening the dispersed repulsive charges and, in the case of divalent ions, the formation of additional salt bridges between negatively charged groups on proteins (Marangoni et al., 2000). For example, when the gels are formed with CaCl2 rather than NaCl, more rigid network is created due to ability of Ca2+ to more effectively screen charges than NaCl and, also, to form ion-bridges by linking negative charges on proteins (Foegeding et al., 2002). The fine stranded, transparent gels are formed by addition of relatively small amounts of salts while turbid and particulate gels are formed due to addition of relatively large amounts of salts (Alting et al., 2003).

Generally acid-induced cold-set gelation is achieved by addition of a food grade acid. Such an acidifier is for example glucono-δ-lactone (GDL) which is hydrolysed to gluconic acid upon addition to water causing a gradual lowering of pH in the medium resulting in gelling at minimum repulsion. Addition of a large amount of GDL results in quick acidification of the medium with the probability of lowering the pH value below the isoelectric point which may cause formation of weaker and brittle gels. On the other hand, the acid-induced gels, formed around the isoelectric point or kept around this pH for extended time periods,

contain larger aggregates and are likely to be stronger and more deformable (Cavallieri and Cunha, 2008). In addition, the amount of GDL required to achieve a certain pH value depends on the protein concentration (Alting et al., 2002).

2.5.1 A general description of proteins and their physicochemical