**3. Characterization of acid milk gels**

#### **3.1 Mechanism of gel formation**

Acid milk gels are typical particle gels where aggregated protein particle forms continuous network structures throughout the entire volume. The mechanism of the gel structure formation is still controversial. Different theoretical models have been proposed to characterize the gel-forming process, including the adhesive hard-sphere model, fractal model, and percolation model [5]. The adhesive hard-sphere model focuses on the surface κ-casein layer of casein micelles. In this model, the highly charged glycomacropeptide (GMP) part of the κ-casein sterically stabilizes the casein micelles [27]. The strong steric repulsion provided by GMP prevented the aggregation of casein micelles against pH and ionic modifications [22, 23, 28]. The rheological behavior of casein micelles fits well with the hard-sphere model [22]. The radius of casein micelles also remains stable at different concentrations. But once the pH approaches the pKa of the charged GMP brush, the surface of casein micelles collapses and aggregates. This model well defined the aggregation behavior of casein micelles. However, it fails to explain the development of gel structure and its mechanical properties [29].

The fractal aggregation model describes that the spherical particles of casein micelles can encounter each other through Brownian motion and form aggregates. The aggregates can then also aggregate with each other. Once no further changes happen among the particles in the aggregate and they are incorporated, this clustercluster aggregation process directs to the aggregates that obey the following scaling Eq. (1):

$$\mathbf{N\_{p}/N\_{0}} = (\mathbf{R/a\_{eff}})\mathbf{D} - \mathbf{3} \tag{1}$$

where Np is the number of particles in an aggregate of radius R, N0 is the total number of initial particles that could form the floc, D is the fractal dimensionality constant (D < 3), and αeff is the radius of the effective building blocks forming the fractal clusters. One limitation of this model is that it assumes all the aggregates have the same size, which is not the case in reality [5]. It also fails to explain the aggregates' rearrangement (before, during, and after gelation), which can influence the D value.

The percolation model combines the concept of fractal aggregate formation and the hard-sphere model. It assumes that percolation clusters form random bonds between adjacent micelles in a lattice, which are random and their sizes increase with the increasing number of bonds. A larger cluster appears above a certain threshold which extends throughout the lattice. Analogies between percolation and gelation can be drawn with particles establishing an increasing number of links as they aggregate until, at a certain threshold, a cluster is created and spans the container/system [2].

*Acid-Induced Gelation of Milk: Formation Mechanism, Gel Characterization, and Influence… DOI: http://dx.doi.org/10.5772/intechopen.107893*

Only a small portion of bonds are joined, and not all individual fractions are incorporated into the system-spanning cluster. This model successfully explained the continuous increase of elastic modulus (G0 ) after gelation [29]. However, it is hard to use this theory to model the mechanical properties of acid gels.

In addition to modeling the acid gelation process, the physical–chemical changes of milk during acidification have been well studied. When the pH of milk is decreased from 6.7 to 6.0, there is a decrease in the net negative charge and reduced electrostatic repulsions. But the solubilization of CCP is minimal, and the micelle integrity is preserved [22]. Decrease the pH from 6.0 to 5.0 leads to further neutralization of the surface charge and shrinkage/collapse of the hairy layer. CCP is fully dissolved, while the internal structure is more homogeneous [3]. At pH lower than 5.0, the destabilized caseins come closer to each other and form the gel structure [4]. Subsequent cooling/ refrigeration causes the gels to swell, increasing the contact area of particles and the gel firmness/strength. As the hydrophobic interaction are lower at low temperatures, the increased gel firmness during cooling storage indicates that other forces, such as electrostatic and van der Waals' interaction, also contribute to the gel integrity [1].

#### **3.2 Rheological properties**

Acid gels are viscoelastic materials. In the dairy industry, the rheometer is the most widely used technique to characterize acid gels. There are two main test methods: small-amplitude oscillatory rheology and large-amplitude oscillatory shear. Large deformation studies can provide information on properties related to the consistency during shearing (a step in the production of stirred-style yogurt) and consumption.

Small-amplitude oscillatory rheology (dynamic testing) is a nondestructive method, involving an applied oscillatory strain or stress that provides very useful information about the gelation process [2, 30]. The main parameters determined during this test include the elastic or storage modulus G<sup>0</sup> , which indicates the energy stored per oscillation cycle, the viscous or loss modulus G″, which indicates the energy dissipated per cycle, and the loss tangent (tan δ), which is the ratio between the viscous modulus and elastic modulus. The definition of these parameters is shown in the following equations:

$$\mathbf{G}' = (\pi \mathbf{0}/\gamma \mathbf{0}) \text{ \textbf{\color{red}{0<}}{\text{cos \textbf{0}}} \text{\textbf{\color{red}{0<}}{\text{g}}} = (\pi \mathbf{0}/\gamma \mathbf{0}) \text{ \textbf{\color{red}{0<}}{\text{sin \textbf{0}}} \text{\textbf{\color{red}{0<}}{\text{tan \textbf{0}}} = \mathbf{G}''/\mathbf{G}' \tag{2}$$

where τ0 is the shear stress, γ0 is the shear strain, and δ is the phase angle.

In reality, the majority of preceding rheological measurements of milk gelation were performed under low strains (<1%) and oscillating strain rates (<0.1 Hz) to avoid gel destruction [7]. The gelation point is where the elastic and viscous modulus cross over (Tan δ = 1) [28]. The rheological properties of acid gel made from unheated milk at 30°C have been well studied, as summarized in previous reviews [1, 2]. After passing the gelation point, the G<sup>0</sup> increased rapidly and plateaued during the aging of the gel. Loss tangent (Tan δ) decreased to <0.4 quickly after gelation and then to around 0.25 during aging. Heat treatment significantly increased the G<sup>0</sup> , and the gelation pH increased from 4.8 to 5.2. [3]. Renan et al. [31] compared the gelation profiles of acid gels produced with culture fermentation and GDL. As shown in **Figure 2**, the elastic modulus of acid gels fermented by culture increased much faster than the GDL. The resulted gels were firmer with a more heterogeneous structure. Both methods produced gels with a similar final loss tangent value of about 0.22. Moreover, acidification methods also influence the rheological properties of acid-

#### **Figure 2.**

*Rheological properties of heat-treated milk during acidification with glucono-delta-lactone at 20°C (black line) or a bacterial culture at 38°C (gray line) in coaxial cylinders versus time. Zero time for bacterial acidification was taken at the time when the temperature reached 38°C. three repetitions for each procedure. (Source: Renan et al. [31]).*

induced gels. The presence of EPS, which are produced by starter culture during fermentation, enhances the protein distribution and viscoelastic properties of acid gels [8, 32].

#### **3.3 Microstructure of acid milk gels**

The microstructure of the gels is directly correlated with their texture, appearance, and organoleptic properties. In the dairy industry, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CSLM) are the most commonly used techniques to observe the microstructure of acid gels. Accordingly, the acid gels consist of a coarse particulate network of casein particles linked together in clusters, chains, and strands [5]. Gastaldi et al. [33] monitored the pH-induced changes of casein micelles during the acidification process. As shown in **Figure 3a**, casein micelles started to aggregate forming clusters when the pH was decreased from 6.7 to 5.8. The initial shape was still discernible. At pH 5.5 to 5.3, most casein particles lost their original structure and were deformed, stretched, and extensively coalesced, forming a pseudo-network with an open structure (**Figure 3b** and **c**). After decreasing the pH to between 4.8 and 4.7, the protein network appeared denser, and the pore size between casein aggregate particles became smaller. At this stage, the formation of acidified milk gels is completed, where the casein particles are aggregated into a threedimensional network (**Figure 3e** and **f**). Much more related research using SEM to investigate the changes in the microstructure of acid gel has been done recently [34, 35]. One shortcoming of SEM is that many preparation steps are required, including dehydration, fixation, embedding, sectioning, and staining, which may disrupt the native structure of gel products and result in the formation of artifacts.

*Acid-Induced Gelation of Milk: Formation Mechanism, Gel Characterization, and Influence… DOI: http://dx.doi.org/10.5772/intechopen.107893*

#### **Figure 3.**

*SEM micrographs of acidified milk critical-point dried samples at different pH: pH 5.8 (a), pH 5.5 (b), pH 5.3 (c), pH 5.0 (d), pH 4.8 (e), pH 4.7 (f). The scale bar represents 1 μm. (Source: Gastaldi et al. [33]).*

Compared to SEM, CLSM is a relatively new technique. It allows observing the overall microstructure of milk gels with minimal preparation steps due to its unique optical sectioning abilities and high spatial resolution [2]. **Figure 4** shows the CLSM images of acid gels produced by GDL or yogurt culture, GDL-produced gel exhibited a denser and more homogeneous structure compared to the gel fermented by culture [31]. Another advantage of CLSM is that it can identify different components in the gel by using specific fluorescence labels. The protein network has been stained with Congo red (0.01% in water) and fluorescein isothiocyanate (FITC, 0.025% in dimethyl sulfoxide) [36]. In another study, the microstructure of low-fat yogurt was observed with CLSM using fast green FCF fluorescent stain to label protein and lectin wheat germ agglutinin Alexafluor 55 conjugate to label EPS produced by starter culture [37]. In reality, the combination of SEM and CLSM can provide more thorough information about the overall and detailed microstructure.

#### **3.4 Syneresis/whey separation**

Syneresis is defined as the spontaneous contraction of a gel, leading to the expulsion of liquid from the pores. In acid milk gel, syneresis is also called whey separation, which refers to the occurrence of whey on the surface of a milk gel. Syneresis relates to the instability of the protein network, which causes a loss of the capacity to entrap

#### **Figure 4.**

*A comparison of the microstructure of acid milk gels produced by yogurt culture (a and c) and GDL (b and d). (Source: Renan et al. [31]).*

the whey in the network [38]. Rapid fermentation, proteolysis, and high incubation temperatures are the main factors that lead to the whey separation of acid gels [39]. Proteolysis during fermentation causes the reduction of interconnections within the protein network and the rearrangement of the intra-network. On the other hand, the acid curds are more prone to syneresis at increased temperatures due to higher rearrangements causing contractions in the gel network, which creates pressure for the whey to move [40].

Whey separation can be simply quantified by determining the quantity of whey expelled from yogurt after centrifugation or drainage through a screen [38, 41, 42]. Both methods are not related to the spontaneous separation of whey from set-style yogurt. The centrifugation method determines the water-holding capacity of the gels under different forces. The drainage of whey from a disrupted gel distributed over a screen measures the whey separation over a large surface area, which is more relevant to the products such as cottage or casein than to set yogurt [2]. Lucey et al. [43] proposed a new method that produces the gels directly in a container and determines the quantity of expelled whey on the surface. During the manufacture of acid-induced gel products, heat treatment is used to sterilize the milk, and gelation is done at a high temperature, which increases whey separation in acid gels. Dairy scientists have used different ways to increase the gel properties, such as the use of high-EPS yield culture [44], enzymatic treatment to strengthen the protein network [3], and increase the protein concentration or adding different exogenous polysaccharides [42, 44, 45].

#### **3.5 Texture properties**

The textural properties of acid milk gels can be measured by different instrumental methods, such as dynamic-amplitude oscillation, large-amplitude oscillatory, texture

*Acid-Induced Gelation of Milk: Formation Mechanism, Gel Characterization, and Influence… DOI: http://dx.doi.org/10.5772/intechopen.107893*

analyzer (penetration), and rotational viscometry [2]. The main challenge for the acid milk gels is the "lumpiness" or "granular" body texture, which is against consumers' expectation of a smooth, fine-bodied product. This textural defect is due to forming large protein aggregates that often range in size from 1 to 5 mm [46]. Many factors contribute to the formation of dense protein clusters, including incubation at a high temperature, rennet, and adding excessive starters [2, 47]. A recent publication indicated that the vibration during fermentation resulted in the formation of bigger aggregates, which caused the graininess of set-style yogurt [48]. In addition, other factors such as a very high amount of total solid and adding excess whey protein concentrate to the milk also increased the "lumpy" or granular defect [2, 49]. Stabilizers, both exogenous and endogenous, have been proved to provide smooth body texture to the acid gel products [44, 50]. Optimizing parameters such as heat treatment, total solid level, amount of additives, amount/variety of starter added, and incubation temperature are necessary to produce acid milk gels with desired texture.
