Functional properties of WPCs: solubility, emulsifying properties and foaming behaviour

The data (Table 3.2) show that the solubility of all 5% (w/w) WPCs at pH 7 was less than 90% with significant (p<0.05) differences among samples and these values were relatively lower than the values obtained in some of the previous studies (Morr et al., 1985: Morr and Foegeding, 1990; Kim et al., 1989; Patel and Kilara, 1990) as well. WP in their native state are highly susceptible to heat induced denaturation (Lee et al., 1992). The reduction in solubility of these WPCs as well as variation in the values may be a consequence of partial protein denaturation due to different processing techniques related to heating and/or drying during concentration. Even partial denaturation of protein during processing may cause flocculation and precipitation during storage which may adversely affect the sensory attributes of the products (Fennema et al., 2008). In addition, the added filtration method carried out to minimise the turbidity interferences with the Bradford assay may have also removed the soluble aggregates of proteins leading to reduction in solubility. Also, WPCs may be modified during manufacturing to improve the functionality (Morr and Foegeding, 1990). The results of particle size analysis (Table 3.2) and especially, non- reducing SDS PAGE (Figure 3.1-B) are useful in understanding the extent of protein denaturation. Smaller particles hydrate more readily resulting in increased solubility (Onwulata et al., 2004; Hudson et al., 2000). The mean particle size of

sample D was significantly (p<0.05) smaller than all others which was further confirmed by results from the non-reducing SDS PAGE. The major protein bands of sample D represented the highest intensity indicating least denaturation of proteins which could observe in particle size analysis as smaller particles.

Sample D also showed appreciably (p<0.05) enhanced solubility. In addition, samples C and F with a higher proportion of native like proteins also exhibited significantly higher solubility and the sample G had a markedly (p< 0.05) larger mean particle size with comparatively low solubility. Sample H had the least solubility around 67%. This sample also showed greater heat stability with fairly high Ca content which contradicted previous reports that indicated enhanced heat sensitivity in the presence of Ca (Morr and Ha, 1993). The average particle size of this sample also was moderate indicating rather preserved native conformation. Therefore this sample was most likely modulated during manufacture in such a way aiming to improve some of the functionalities. On the other hand this modulation could have interfered with the solubility measurements of WPC using Bradford reagent. Additionally, these WPCs contained considerable amounts of fat. Bound fat may also alter the protein water interactions thus decreasing the solubility (Patel and Kilara, 1990) and bringing discrepancies among methods used in protein content determination (Morr et al., 1985).

Figure 3.1 The reducing (A) and non-reducing (B) SDS-PAGE of 8 commercial WPC samples. Molecular weight markers (MW Markers); β-lactoglobulin (β- Lg);

Figure 3.2 Proportion of major proteins present in 8 commercial WPC samples (WPC A - H) as detected by size exclusion HPLC. Different letters indicate significant difference (p<0.05) among samples within a particular protein type.

Table 3.2 Colloidal, interfacial and gelling properties of commercial whey protein concentrates

Product Solubility, % Particle size, nm EAI, m2.g-1 ESI, h Adsorbed protein mg.mL-1 Overrun, % Gel firmness g mm.s-2 WHC, % A 81.0±0.8ab 197.3±7.3cd 6119±171a 23.8±0.1a 54.6±0.6d 296±4.1b 664±126.9cd 98.8±0.1e B 78.7±1.7ab 197.0±6.7cd 4924±966a 23.7±0.1a 61.3±0.8b 336±2.4a 1296±103.0c 99.5±0.1cd C 86.5±4.4a 214.1±4.4b 5136±909a 23.8±0.1a 55.3±2.3cd 0d 872±23.6cd 98.9±0.2e D 86.3±2.5a 185.1±4.3d 5229±837a 23.7±0.1a 53.3±0.6de 341±3.1a 2955±507.5a 99.5±0.1d E 80.6±6.8ab 205.1±12.0bc 6240±493a 22.8±0.9a 65.0±1.8a 0d 523±162.3d 99.7±0.1bc F 83.2±4.7a 209.8±7.5b 5597±484a 23.0±1.3a 58.9±1.9bc 226±5.7c 240±496.7ab 100.0±0.0a G 74.0±1.9bc 229.6±10.6a 4788±203a 23.6±0.3a 50.9±0.8e 0d 453±62.7d 99.4±0.1d H 66.9±2.3c 203.0±6.4bc 5337±369a 22.9±1.5a 50.3±1.5e 231±14.8c 2043±277.0b 99.9±0.1ab

A, B, C, D, E, F, G, H – commercial whey protein concentrates (WPCs); EAI – emulsion activity index; ESI – emulsion stability index; WHC – water holding capacity. Means present the average of at least4 independent observations (n=4). Means with different superscript letter are significantly different. (P < 0.05).

Emulsifying activity measures the turbidity at a single wavelength to assess the droplet size and, by implication, the interfacial area of the emulsion (Lee et al., 1992). The capacity of protein to stabilize an emulsion may be related to the interfacial area. Also, the stability of an emulsion is related to the consistency of the interfacial area (Pearce and Kinsella, 1978). The four main destabilization mechanisms related to dispersions of protein-coated oil droplets are creaming, coalescence, Ostwald ripening and flocculation. Among them, the most complicated one to control is droplet flocculation which can be delicately affected by the other factors and also it can influence directly the emulsion structure, and other stabilising mechanisms, especially creaming and coalescence. Also, the physico-chemical principles governing stability are depending on the classical electrostatic and steric stabilisation. Electrostatic stabilisation is governed by the presence of electrical charges on the surface of the droplets, and the stabilising layer absorbed at the droplet surface. The greater the surface charge density and the lower the ionic strength of the aqueous medium, the more stable is the emulsion. Steric stabilisation arises from the presence of a polymeric or the steric obstruction at the droplet surface. More importantly, for the long-term stabilisation, the adsorbing polymer must be present at a sufficient concentration to cover the oil-water interface completely the adsorbed layer must remain permanently attached to the interface with parts of individual molecules projecting away from the surface towards the aqueous medium. Also, the steric and electrostatic contributions depend on the specific protein structure and the pH of the solution, for example, the contribution of electrostatic stabilisation is generally greater for adsorbed monolayers of compact globular proteins at pH values well way from the isoelectric point (Dickinson, 2010). In addition, net hydrophobicity of proteins, solubility and composition of WPC are correlated with emulsifying ability (Klemaszewski and Kinsella, 1991). However, as these results show all samples did not exhibit (Table 3.2) any significant difference in EAI and emulsion stability.

Proteins contribute to film formation in foams by concentrating at the interface which depends on the ability of diffusion, reducing interfacial tension and partially unfolding. The covalent disulfide bonds and secondary forces are important in reacting with neighbouring protein molecules to form continuous cohesive films. In properly homogenised protein systems, the principal attractive forces between continuous proteins are short-range hydrogen bonds, hydrophobic and electrostatic interactions and van der Waals forces as well. The ionic strength, temperature and the pH of the medium which affects the partial protein denaturation as well as protein concentration which may alter water structure with subsequent changes in hydrophobic interactions (Phillips et al., 1991; Kinsella, 1984) are equally important in forming properties. In addition, the foaming properties of WPC depend on the degree of denaturation, calcium ion concentration and lipid content (Jovanovic et al., 2005). Moreover at pH 7 whey proteins have a net negative charge which favours foam formation by enhancing protein unfolding via activated thiol groups (Lee et al., 1992). In general free fat and bound fat were negatively related with foaming properties, where as ash, calcium content positively related to (Patel and Kilara, 1990).

As shown in Table 3.2, several WPC samples (C, E & G) failed to create foam at all. Furthermore all samples exhibited low foam stability. Impaired foaming capacity implies the inability of protein to incorporate air in the liquid continuous phase and to produce strong, cohesive and stable films around air bubbles. Proximate analysis of WPCs indicated that all samples contained considerable amount of fat. Mangino et al., (1987) have shown that WPC prepared from pasteurized milk gives higher foam overrun and stability and that this is probably related to a decrease in neutral lipids during pasteurization.Lipid content in WPC can seriously impair the foaming process since surface active, polar lipids interfere with protein films by situating themselves at air/water interface (Fennama, 1996), and weaken the foaming properties by inhibiting adsorption of protein to the interphase. Furthermore, these interfering substances possess weak

cohesive and visco-elastic properties to overcome the internal pressure of air bubbles compared to whey proteins resulting rapid collapsing of bubbles.