**4. Strategies to improve the acid-induced milk gels**

#### **4.1 Heating**

Heat treatment is a standard procedure before further processing of the dairy product. In the production of fermented dairy products such as yogurt, the heat treatment is usually performed at high temperatures (such as 90°C, 5 min). Heating can destroy the raw milk flora and decrease the dissolved oxygen level which can prevent the growth of the starter cultures. More importantly, heat treatment can denature the whey proteins in milk, increasing the firmness and texture of acid milk gels [3, 51].

The structure of casein micelle is relatively heat-stable as they lack tertiary structure. On the contrary, the main globular whey proteins such as α-lactoalbumin (α-la) and β-lactoglobulin (β-LG) undergo irreversible denaturation at temperatures higher than 70°C [52]. The denatured whey proteins can aggregate with themselves or free caseins (mainly dissociated κ-casein) through disulfide bonds and hydrophobic interaction [53]. Denatured β-LG can also attach to the surface of casein micelles by interacting with the surface κ-casein layer. The coating of denatured whey proteins is pH-dependent. At neutral pH 6.7, heating resulted in around 30% of denatured whey proteins associated with the surface of casein micelles, and this number increased to 75% when heating at pH 6.3 [54]. Whey protein denaturation significantly altered the acid gelation behavior of milk, particularly at a higher denaturation degree (>40%). The gelation pH increased from 4.9 to values between 5.1 and 5.3, and the elastic increased drastically [55].

The distribution of denatured whey proteins between serum and the surface of casein micelles has a significant influence on the gelation process. At lower pH 6.3, most denatured whey proteins are present on the surface of casein micelles, and they gel first entrapping the casein micelles and triggering gelation at pH 5.3. In contrast, at pH 7.0, most denatured whey proteins are present in the serum phase as soluble aggregates, which contribute to the formation of stiff gels by associating with casein micelles during acidification [51]. Both heating at lower pH (<6.7) and higher pH (>6.7) resulted in slightly weaker acid gels than at neutral pH (6.7) [56–58].

There are still some divergent opinions regarding the role of soluble whey protein complexes and micelle bind complexes in the acid milk gels. Some researchers think that the small number of denatured whey proteins associated with casein micelles

during heating is responsible for the increased gel properties [43, 58]. In contrast, other researchers think that the soluble denatured whey proteins play a more crucial role than the denatured whey proteins associated with casein micelle [56, 59]. In recent research, glutaraldehyde was added to milk to reduce micellar kappa-casein dissociation, which decreased the formation of soluble protein complexes. This reduction in soluble complexes resulted in the forming of weaker gels [60]. In addition to the gelation process, high heat treatment increases the brittleness of acid gels prepared by microbial fermentation, while it decreases the brittleness of gels prepared via GDL [51, 61]. The differences are due to different acidification rates between these two methods.

#### **4.2 High-pressure treatment**

High-pressure treatment can reduce milk fat globule size, disintegrate/re-associate casein micelles, and denature whey proteins [4]. The size of casein micelles is stable under pressures lower than 200 MPa [62]. Increasing pressure to 250 MPa increased micelle size by 25%, whereas a further increase of pressure (300–800 MPa) decreased casein micelle radius by about 50% [63]. The soluble caseins and soluble calcium increased after high-pressure treatment [64].

High-pressure treatments significantly improve the acid coagulation behavior of milk. The rigidity, strength, and resistance to syneresis of acid gels were improved [13, 65, 66], which are a result of the increases in protein hydration and density of network strands, resulting from the incorporation of denatured whey proteins in the acid gel [13, 67]. The elastic modulus and yield stress of acid milk gels increased with decreasing fat globule size as the adsorption of proteins onto the newly created surface of fat globules after high-pressure treatment, resulting in the formation of a more porous protein network with thick strands [66]. Homogenization performed prior to heating resulted in higher adsorption of proteins to the fat globules than homogenization after heating, which further led to the formation of acid gels with higher elastic modulus and yield stress [68].

#### **4.3 Ultrasonication**

Ultrasound refers to sound waves with a frequency higher than 20 kHz, which modifies the structure functionality of protein molecules through the cavitation effect, based on the implosion of bubbles that produce shock waves surrounding the probe and jets of high velocity. It is a relatively new technique used in dairy processing to improve the acid gelation properties of milk. The influence of ultrasound on the lactic fermentation, growth and cell viability of lactic acid bacteria, lactose metabolism, texture, and sensory attributes of fermented dairy products has been reviewed recently [69, 70].

Pretreatment of caseins with ultrasound postponed the gelation point to lower pH, decreased the syneresis, and enhanced the elasticity of acid gels, which have a more interconnected structure [71, 72]. The increased acid gelation properties are related to the increased surface hydrophobicity [73]. Whey protein denaturation and increased association of casein with the milk fat globule membrane during ultrasonication also contribute to the increased gel strength [74]. In contrast, ultrasound treatment during the lag phase of lactic acid bacteria reduced the fermentation time, promoted the speed of lactose hydrolysis, and increased the storage modulus of the final gels [75]. For the yogurt products with high protein concentration, ultrasonication during

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

fermentation (for instance from pH 5.8 to 5.1) decreased the firmness and provided a smooth texture for yogurt products, which solved the difficulty of further processing issues [76]. The influence of ultrasonication on acid gelation properties is temperature-dependent. Ultrasonication at temperatures lower than 60°C produced acid gels with higher firmness than those produced at temperatures higher than 60°C [77].

#### **4.4 Enzymatic treatment**

Rennet is a complex of enzymes with the active enzyme chymosin, which works on the Phe105-Met106 bond of surface κ-caseins. It cuts κ-casein into hydrophobic para-κ-casein, which remains on the surface of casein micelle, and hydrophilic GMP, which is cleaved from casein micelle [3]. Partial hydrolysis of κ-caseins reduces the negative charge of casein micelles, promoting aggregation during acidification [78]. The gelation pH and elastic modulus increased with increasing hydrolysis degree [79, 80]. Inactivation of enzymes at a lower temperature (60°C) resulted in firmer acid gels than at a higher temperature (85°C). However, gels produced after partial κcasein hydrolysis exhibited higher syneresis [80].

Transglutaminase (TG) is another enzyme used to improve the acid gelation behavior of milk. It crosslinks peptides and proteins through an acyl transfer mechanism between glutamine and lysine residues [81]. At neutral pH, TG predominately works on the κ-casein surface layer of casein micelles, which prevents the dissociation of κ-caseins, and increases the colloidal stability of casein micelles [82]. TG treatment positively influences the physical properties and microstructure of the yogurt gels [83]. It prevented the release of the caseins into the serum phase which further decreased the formation of soluble complexes during heating [84, 85]. The rearrangements within the protein network were also limited by TG during the gelation, which produced acid gels with a more homogeneous network consisting of smaller aggregates and better WHC [83].

#### **4.5 Endogenous and exogenous polysaccharides**

Exopolysaccharide (EPS)-producing starter cultures are preferred in the manufacture of fermented products. The production of EPS in situ has been shown to improve the texture and rheological properties of the yogurt [46, 86]. EPS can improve the structure of milk gels by attaching to the protein network and the bacteria and forming a web-like structure [87]. The influence of EPS on the physical properties of acid gels are affected by EPS location, its structure (molecular mass, side chains, stiffness, and charge), and the interactions of EPS with other components (proteins and minerals) [88]. Depending on the location, EPS can be divided into ropy-EPS (free EPS in the medium) and capsular EPS (located on the surface of the bacterial cells) [89]. Ropy-EPS can produce a stringy and slimy appearance and affect the rheological properties and microstructure of milk gels. Most EPS-producing strains can increase the firmness and WHC of acid gels compared with non-EPS strains. However, slightly weaker gels produced from EPS-producing strains have been reported [90]. Charged EPS can interact with milk proteins through electrostatic attractions during fermentation which increases the gel texture, whereas uncharged EPS can induce depletion flocculation in casein systems [91].

A wide variety of endogenous polysaccharides have been used as additives in the production of acid milk gels in recent years. They can combine with water in the gel and interact with milk proteins during fermentation and storage, resulting in the formation of gels with improved texture and sensory properties. When selecting polysaccharide additives, many factors need to be considered, such as structure, charge properties, and adding amount. Pang et al. [92] found that anionic polysaccharides enhanced the acid gelation properties of yogurt. In contrast, neutral polysaccharides inhibited milk gelation from the beginning [92]. Apple pomace (pectin and soluble fibers) improved the firmness and cohesiveness of set yogurt. At the highest adding amount of 1%, gelation happened at a much higher point (pH 5.9) [93]. Many other polysaccharides, such as okra polysaccharide, dietary fiber, salecan, and oat β-glucan, have been used to improve the structure and rheological properties of acid milk gels recently [44, 94–96].

#### **4.6 Functional bioactive compounds**

Phenolic compounds can combine with milk proteins through hydrophobic interactions [4, 97]. Polyphenol addition does not influence the fermentation process or the lactic acid bacteria viability during the storage of yogurt [98]. Because of the capacity of polyphenols to interact with milk proteins, the acid gels incorporated with phenolic compounds had a higher firmness value and a stronger water-binding capacity within the gel matrix [99–101]. The influence of phenolic compounds on gelation and the physical properties of acid milk gels depends on the source and addition timing. Phenolic compounds extracted from different herbs (thistle, hawthorn, marjoram, and sage) prevented the syneresis and improved the water-holding capacity of yogurt [102]. Polyphenols extracted from the honeysuckle berries did not influence the rheological properties of yogurt but decreased the viscosity during storage [103]. In contrast, EI-Said et al. noticed that adding pomegranate peel extracts to milk decreased the viscosity of stirred yogurt [104]. The addition of gallic acid before heat treatment resulted in a longer gelation time and decreased final storage modulus (G<sup>0</sup> ) and fracture stress. On the other hand, no influence was found when adding gallic acid after heat treatment [105].
