**2. Techno-functional properties of milk proteins**

### **2.1 Foaming properties**

Milk is well known by its important foaming properties encountered with many various milk-based aerated foods such as ice cream, cappuccino, whipped cream, chocolate mousse, etc. [4]. Indeed, milk proteins determine the structure and stability of milk foam and emulsions due to their particular physicochemical characteristics as well as their interaction with other milk constituents [4–6].

Foaming properties of milk proteins are attributed to their ability to:

1. absorb at the air-water interface leading to a rapid decrease of surface tension at the air-water interface

**Figure 1.** *Schematic presentation of protein based foam.*


According to their structure and surface rheological properties, milk proteins can be classified in two main groups [8–10] flexible and globular proteins:


All milk proteins (β-casein, α-casein, κ-casein, β-lactoglobulin and α-lactalbumin) compete to the interface as follows: proteins with a more flexible structure such as β-casein are quickly adsorbed, whereas, globular proteins adsorb slowly [11]. Hence, the β-casein causes the creation of the foam due to its disordered structure. Indeed, it is considered as a "mobile" protein with an intrinsically unstructured molecular structure [12]. On the other hand, despite the low adsorption of whey globular proteins (β-lactoglobulin and α-lactalbumin), they intensively contribute to the formation of the protein film by improving its rigidity [13]. The order of the foaming efficiency of the insoluble and soluble protein fractions respectively of cow's milk is as follows: β casein> α casein = κ casein> whole casein, β-lactoglobulin> α-lactalbumin> whey [14].

Finally, whey globular proteins are characterized by a lower ability to adsorb at interfaces than those of caseins. On the other hand, their compact structure stabilized by the disulfide bridges, makes them suitable for creating a rigid interfacial protein film and consequently a higher ability to stabilize foams [8, 15].

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

The foamability of purified whey proteins is higher than that of the whole extracted whey. The β-lactoglobulin is the predominant adsorbed protein on the interface, regardless of its concentration ratio with α-lactalbumin [14]. At pH 6.7, this protein exists as dimers which are maintained by non-covalent interactions. Each monomer is characterized by two intramolecular disulfide bridges and a free thiol group. Upon adsorption at the interface, the β-lactoglobulin is not fully unfolded and its rate of lowering interfacial tension is slower compared to that of β-casein. However, once adsorbed, the created protein film of β-lactoglobulin is distinguished by a high density and an important protein–protein interaction in comparison with the protein layers of caseins. Indeed, the partial unfolding of β-lactoglobulin during its adsorption at the interface leads to the exposure of its free thiol group. Consequently, the adsorbed protein undergoes slow polymerization which is explained by the exchange between free thiol groups and disulfide bridges between the adsorbed β-lactoglobulin dimers [12, 16].

The purified β-lactoglobulin showed a better tensioactivity compared to other whey proteins such as the α-lactalbumin [17]. The β-lactoglobulin is characterized by significant foaming and stabilizing properties due to its high hydrophobicity and its unstructured conformation. On the other hand, the α-lactalbumin has interesting foaming properties but a low foaming stability [18]. This behavior is attributed to the compact globular structure of α-lactalbumin and the presence of four buried disulfide bridges which reduce its flexibility, and therefore its foaming and emulsifying properties [19].

Brooker et al. [20] showed that the main constituents of the milk foam interface are β-casein, β-lactoglobulin and α-lactalbumin. Other studies have shown that the stability of milk froth increases with increasing β-casein content [4, 10, 21, 22]. Indeed, during the creation of dairy foams, the β-casein is first adsorbed protein on the interface with a faster diffusion than that of globular whey proteins [23]. Thus, β-casein, once injected into a casein solution, is even able of moving other caseins such as αS1-casein and β-lactoglobulin from the interface, while the reverse phenomenon is difficult to achieve (**Figure 2**) [25].

Thus, β-casein plays the key role in the stabilization of the foam due to its wellstructured molecular conformation. It is even able to dissociate the αS1-β complexes releasing the αS1-casein and β-casein monomers. This behavior can be observed only at pH levels above 6, indeed at a pH close to 4.5, the solubility dominates foaming properties of caseins regardless of pH value [17].

Bovine proteins mixtures (β-casein-β-lactoglobulin and β-casein-α-lactalbumin) at different mixture ratios (100,0, 75:25; 50:50; 25:75; 0:100) presented an intermediate foaming behavior between those of pure β-casein and globular proteins alone (α-lactalbumin or β-lactoglobulin): the added β-casein increased significantly the foaming capacity value of protein solution. For β-casein-α-lactalbumin mixture, an increase of β-casein proportion from 25–75% of total protein amount, significantly increased foamability of 41%. For β-casein-β-lactoglobulin protein mixture, the foamability of the mixed systems was mainly dominated by β-casein. For instance, foaming capacity increased of 46.2% between pure β-lactoglobulin and the mixture

**Figure 2.**

*The incorporation of casein in the structure of the* β*-lactoglobulin adsorbed layer; (a) monolayer of*  β*-lactoglobulin; (b) incorporation of caseins into the* β*-lactoglobulin layer [24].*

containing 50% of β-casein and 50% of β-lactoglobulin [10]. On the other hand, the foam stability is mainly governed by the β-casein regardless of the other mixed protein (β-lactoglobulin or α-lactalbumin). Indeed, the increase in the stability of foams is attributed to an increase in the diffusion and adsorption of milk proteins at the airwater interface [10, 26]. In the same way, Xiong et al. [27] studied foaming properties of caseins: whey proteins mixture at different ratios (80:20–75:25 and 80:20–40:60). These authors found that proteins at a ratio of 40:60 exhibited the lowest foam stability compared to that of 80: 20 sample because of the adsorption and spreading behavior of micellar caseins at the air-water interface, whereas, samples with ratios 80:20 and 75:25 did not show any significant difference in foaming properties [27].

Laleye et al. [28] reported that the milk origin and consequently the protein composition of whey have a great influence on its foaming and emulsifying properties. For instance, bovine and camel whey presented different foaming properties, which is attributed to the difference in protein composition of both wheys especially the absence of β-lactoglobulin in camel milk.

### *2.1.1 Effect of pH on foaming properties*

Milk proteins molecules change their conformation and surface activity depending on pH level. Hence, foaming and interfacial properties also change depending on the physicochemical parameters of proteins [8]. For instance, foaming properties of skimmed milk decrease considerably at acidic pH (pH 4–5) because of caseins precipitation. However, these properties increase at pH 3 due to the dissociation of the casein micelles and the re-solubilized caseins characterized by a higher tensioactivity [23].

Surface properties of caseinates are predominantly determined by the β-casein regardless of pH value. Furthermore, surface pressure isotherms of caseinates were nearly identical to those of pure β-casein. Hence, caseinates adsorption layers were modeled by treating them as β-casein ones [8, 11]. The β-casein polypeptide is constituted of 209 amino acid residues; the first 50 are mainly hydrophilic, while the remaining 159 residues are mainly hydrophobic [29].

Neutron reflectivity studies [9, 30] have shown that the adsorbed β-casein layer can be represented as a dense inner layer adjacent to the interface with a thickness of 1–2.5 nm and another less dense outer layer released in the aqueous phase 3–7.5 nm in length. The inner layer includes the hydrophobic amino acids in a "train" configuration, while the outer layer is extended as a "tail" or "loop" constituting of hydrophilic amino acids. These data were used by Marinova et al. [8] in order to schematize sodium caseinates adsorption behavior at the air-water interface (**Figure 3a**). By reducing the pH to the pI (Isoelectric pH) of β-casein, the hydrophilic residues are electrically neutral at this pH value resulting a decrease the thickness of the protein layer (**Figure 3a**). Consequently, the decrease in the foaming properties of β-casein is caused by the precipitation proteins leading to a lower protein coverage of interface and a reduced electrostatic repulsion between protein films [8].

Unlike the foaming and interfacial properties of sodium caseinates, whey foams more at a pH levels close to the pI of β-lactoglobulin (pI = 5.2) and α-lactalbumin (pI = 4.1–4.8). At this pH value, the foam created by whey is more stable than that at neutral pH due to the reduced negative charge and electrostatic repulsion of proteins [5, 8, 23]. The modeling of whey protein adsorption layers is not realized by the major protein alone (β-lactoglobulin) as observed for sodium caseinates. Marinova et al. [8] represented the adsorbed layer of the whey protein mixture by an "average" of globular proteins which adsorb almost intact at the interface. At neutral pH, the molecule is negatively charged and electrostatic repulsions prevent the formation of a dense and continuous protein adsorption layer.

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

**Figure 3.**

*Schematic presentation of caseinates (a) and WPC (whey protein concentrate) (b) at air-water surface at neutral pH (~7) and isoelectric pH (~4.5) [8].*

However, in acidic conditions, the molecules are not charged and their adsorption and interaction are much higher (**Figure 3b**).

At pH 6,7, Lajnaf et al. [15] showed that the adsorbed protein layer of whey at the air-water interface consists of the β-lactoglobulin, while at pH 4.6, the adsorbed protein layer consists of the α-lactalbumin which is the most surface active protein in whey in acidic conditions. Indeed, the α-lactalbumin loses its bound calcium ion at pH values less than 5 and takes on the molten globular state and hence, becomes more surface active. However, the β-lactoglobulin is more rigid and thermodynamically stable at low pH levels leading to a less competitive adsorption of the protein in acidic conditions [23, 30–32].

### *2.1.2 Effect of temperature on foaming properties*

Temperature is a very important parameter which affects the conformation of milk proteins and their distribution between both of whey and the colloidal phases of milk [33]. Therefore, temperature affects the molecular structure and foaming properties of milk proteins [33, 34].

Foaming properties of milk are significantly enhanced by increasing the temperature from 45–85°C, whereas stabilizing foam ability are maximum at 45°C [35]. After heating at 50°C, transmission electron microscopic observations shows that the film protein at the air-water interface consists mainly of the soluble caseins as well as whey proteins [4].

Overall, the denaturation of milk proteins after thermal treatments improves their foaming and interfacial properties due to their increased molecular flexibility, as well as their surface hydrophobicity [36]. However, foaming behavior heated milk proteins usually depends on the rate of protein aggregation. Denaturated and unaggregated proteins adsorb faster at the interface than aggregates, leading to the creation of foam. On the other hand, the adsorption of aggregates is slower, whereas, they contribute to the stability of the created foam (**Figure 4**) [37, 38].

Furthermore, greater foaming and stabilizing properties was measured for bovine milk proteins after increasing the temperature of thermal treatments, (up to 90°C for 30 min). This behavior was linked to the heat denaturation and aggregation of milk proteins especially globular whey proteins (β-lactoglobulin and α-lactalbumin), which led to an increase in the surface hydrophobicity and a decrease in the electronegative charge and interfacial tension [39].

**Figure 4.**

*Schematic representation of milk protein adsorbed layers adsorbed at the air-water interface by mixing unaggregated proteins and aggregates that within a heat treatment [38].*

Similarly, whey proteins improve their foaming and stabilizing properties after heating process. However, the excessive heating denaturation of leads to a reduction of the resulted foam volume (for instance: 85°C for 750 s). Heating improves the tensioactive properties of α-lactalbumin and β-lactoglobulin by the exposure of the buried hydrophobic molecular parts of proteins leading to an improvement in their foaming and emulsifying properties [14].

## **2.2 Emulsifying properties**

Emulsification is a common operation in food industry which is encountered with various food products such as mayonnaise sauces, soft drinks, salad dressings, soups, creams, butter and margarine [40]. Overall, an emulsion is obtained by mixing two immiscible liquids in the presence of one or more emulsifiers, where one is finely dispersed as droplets within another as oil in water emulsions (**Figure 5**) [16, 41]. During homogenization, emulsifiers are adsorbed onto the interfaces of freshly formed oil droplets leading to the reduction of the interfacial tension and oil droplets disruption. The most common emulsifiers used in the food industry are proteins which are the most surface-active agents in formulated emulsion systems [42].

During emulsion creation, mechanical shear is induced to create oil droplets within a continuous aqueous phase. Proteins dissolved onto this phase migrate to

### **Figure 5.**

*Microscopic images of oil-in-water emulsions (85%) stabilized by whey protein isolate emulsion. The emulsion is diluted in a solution of SDS 0.1%.*

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

the interface, and then realign to position its hydrophilic and hydrophobic amino acids towards water and oil phases, respectively. Once adsorbed, proteins accumulate to form a viscoelastic film around the created oil droplet and to keep the emulsion stable [41, 43].

Caseins are well known by their ability to adsorb rapidly at the oil–water interface, they are more effective in decreasing the interfacial tension than whey proteins. Furthermore, all casein types are adsorbed at the surface of oil droplet to provide stability to the resultant emulsion against coalescence and flocculation [31, 44]. Previous works evidenced that the diffusion and reorientation of β-casein at the interface occurs more rapidly than β-lactoglobulin and α-lactalbumin due to the low structuring of β-casein. Indeed, the β-casein is a flexible protein characterized by an amphiphilic nature allowing it to be the most effective in reducing surface tension at the oil–water when compared to β-lactoglobulin and even whole milk [12]. The β-casein is considered as a "disordered mobile protein" due to the low structuring molecular conformation and its rapid diffusion at the oil–water interface. It can occupy the majority of interfacial sites leading to a complete or partial replacement of the β-lactoglobulin molecules from the interface [45]. Seta et al. [45] noted that the protein mixtures containing different proportions of β-lactoglobulin and β-casein (1:3, 1:1 and 3:1) at pH 6.8 had an interfacial behavior similar to that of pure β-casein, suggesting the dominance of β-casein at the oil–water interface. Assessment of *in vitro* digestibility of milk protein isolate showed reduced emulsion stability compared with the intact proteins emulsions. Emulsion instability was hydrolytic enzyme preparation dependent and increased with increasing the degree of hydrolysis for a given enzyme [46].
