*2.2.1 Effect of pH on emulsifying properties*

Emulsifying properties of milk proteins change significantly depending on pH level of proteins [41]. Mellema and Isenbart [47] studied the effect of acidification of a solution of reconstituted skim milk powder and whey protein on their interfacial properties (at a concentration of 0.7% (w/w)). These authors found that at pH 4.6, acidified casein micelles lose their colloidal stability, they aggregate and become less amphiphilic and tensioactive. Unlike the foaming and interfacial properties of sodium caseinates, whey proteins improve their flexibility when lowering pH level from 6.7 to 4.6. The dominant whey protein at the oil–water interface in acidic conditions is the α-lactalbumin: this protein adsorbs slowly at the interface but gives a high viscoelastic modulus [47]. The β-casein coated and stabilized the oil-droplets better at pH levels above neutrality when compared to acidic conditions. However, emulsions made camel β-casein at pH ~ 5 were unstable leading to significantly bigger oil droplets. Indeed, the acidification of caseins usually leads to the decrease in emulsion activity and stability because of precipitation and aggregation which alter their amphiphilic nature [42].

For whey proteins, Kilian et al. [48] compared their emulsifying behavior in both pH values 5.7 and 7.0. These authors reported that the emulsion was more stable in pH 5.7 than that at pH 7.0 with lower diameter droplets. However, the interfacial film formed by the proteins presented an essentially elastic behavior in both pH values with no significant differences in the resistance parameters of the oil–water layer interface [48]. Lam and Nickerson [19, 41] found that EAI (Emulsion Activity Index) as well as ESI (Emulsion Stability Index) values of whey protein isolate and the pure α-lactalbumin declined when pH increased from pH ~ 3 to pH ~ 5, before increasing at pH ~ 7. Stability of emulsions depends on the charge of the proteins: a higher stability is observed under conditions where electrostatic repulsion occurs. Indeed, electrostatic repulsion aided in keeping droplets from

flocculating. However, this behavior was less effective for neutrally charged protein near its pI [19]. Emulsifying properties of whey protein aggregates were also investigated. These fabricated aggregates (native, nanoparticles, and nanofibrils) showed significant emulsifying properties at pH 3 especially for whey nanofibrils. However, whey proteins nanoparticles had the highest EAI and ESI values at neutral pH [49].

The results of Lajnaf et al. [50] indicated that the α-lactalbumin molecules at neutral pH coated the oil-droplets better than those in acidic conditions with higher EAI values of apo bovine α-lactalbumin proteins (without calcium). Furthermore, ESI values of both apo and holo (with calcium) states of the α-lactalbumin were higher at pH ~ 7 than those at pH ~ 5. This behavior was explained by the electrostatic repulsive forces of the α-lactalbumin far from its pI which led to a better adsorption of the protein to the oil-droplet surface [50–52].

### *2.2.2 Effect of heating temperature on emulsifying properties*

Structure–function relationships of heated milk proteins has been widely studied in the literature, especially as it relates to their aggregative properties after heating and nature of interactions (thiol-disulfide exchange reactions, hydrophobic interactions, and electrostatic interactions hydrogen bonding) [19, 39, 41, 53, 54]. These interactions can even alter the physicochemical and emulsifying properties of milk proteins molecules by heating the proteins to a partial or complete denaturation of the protein structure and to expose buried hydrophobic moieties [41].

The surface protein coverage of emulsions created with heated calcium caseinates solutions at 121°C for 15 min was higher compared to that of native caseinates. This behavior was attributed to protein aggregation upon heating and to the higher viscosity of the aqueous phase. On the other hand, milk proteins heating induces the increase in emulsion stability due to an increase in the diffusion and adsorption velocity of milk proteins at the interface and a decreased apparent viscosity [26, 44]. On the other hand, the emulsifying properties of whey protein were strongly associated with the size of generated thermo-induced aggregates [41]. For instance, heated whey proteins at 85°C and at pH 7 exhibited lower emulsifying compared to those heated at 55°C and 25°C. The difference in the size of the aggregates as a function of temperature: larger aggregates are usually obtained after heating at a higher temperature. Furthermore, Lam and Nickerson [41] found that EAI values of whey protein isolate were greater at both pH 3 and 7 since protein aggregates are smaller and the

### **Figure 6.**

*Schematic presentation of whey protein based emulsion after a thermal treatment of proteins at neutral pH (a) and in acidic conditions (b).*

*Promising Food Ingredients: Milk Proteins DOI: http://dx.doi.org/10.5772/intechopen.99092*

hydrodynamic radii of the generated aggregates are lower leading to a rapid migration and integration of heated proteins into the interface (**Figure 6a**). In contrast, protein–protein aggregation was the highest after heating whey proteins at acid pH resulting a reduction in their EAI values. Indeed, the aggregation and hydrodynamic radii of the whey protein aggregates were highest in these conditions because of the reduction in electrostatic repulsion between heated proteins close to their pI (**Figure 6b**).

For pure whey proteins, the applied heat treatment to the α-lactalbumin at 65°C improves its stability to create and stabilize emulsions when compared to the unheated α-lactalbumin. However, increasing the temperature of the heat treatment from 65–95°C for 30 min leads to in reduction in its emulsifying stability because of the excessive denaturation of this protein [19].
