**2. Acidification of milk**

#### **2.1 Method of acidification**

There are different methods to acidify milk, including the direct addition of acids, such as lactic acid [15], citric acid [16], citric acid, and sulfuric acid [17], and indirect fermentation by bacterial culture or glucono-delta-lactone (GDL) which hydrolyses into gluconic acid in solution [3]. The addition of inorganic acid can decrease the pH of the milk rapidly, while indirect fermentation decreases the pH slowly. The hydrolysis of GDL is temperature-dependent, where the pH reduction is more rapid at a higher temperature. GDL decreases the pH much faster initially than culture but then stabilizes. By contrast, the pH of milk added with starter bacteria continues to decrease slowly with time. The final pH of GDL-induced gel is determined by the amount of GDL added to the milk, while the pH of culture-induced gel can reach a very low pH (e.g., 4.1) until the bacterial activity is inhibited [5]. The differences in

the acidification rate lead to different changes in the physicochemical properties of casein micelles and the aggregation behavior of casein particles, which further influence the rheological and physical properties of the final acid gels.

#### **2.2 Structure of casein micelle and its changes during acidification**

The formation of a gel structure of milk is mainly from the changes in milk proteins, particularly for the caseins, which constitute approximately 80% of total milk protein. There are four main caseins: αS1, αS2, β, and κ-caseins with a ratio of 4:1:3.5:1.5 [18]. Caseins cannot form a globular structure due to the presence of a high amount of proline [3]. Alternatively, caseins can combine with calcium and assemble into a particular spherical micellar structure, named casein micelles, which have a diameter range of 50–500 nm (average 150 nm), containing 94% protein and 6% minerals (calcium, phosphate, magnesium, and citrate) [19]. The structure of casein micelle has been developed over the past decades. Among all the proposed models, the nanocluster model proposed by Holt et al. [3] can best characterize all phenomena that occurred to milk during processing. This nanocluster model was improved by Dalgleish and Corredig [18], considering the location of a large amount of water in the micellar interior, as shown in **Figure 1**.

In the nanocluster model, αS- and β-caseins (blue coils), which are rich in phosphoserine in their structure, are considered to interact with and surround the colloidal calcium phosphate nanoclusters (black spheres), forming the internal structure of casein micelles through hydrophobic interaction and hydrogen bond [4]. κcaseins lack phosphate centers and are present on the surface providing strong steric repulsion to maintain their colloidal stability [20, 21]. Casein micelles have an isoelectric point of 4.6.

Acidification influences both the surface and internal structure of casein micelles. The colloidal calcium phosphate gradually dissociates from casein micelles [22], the surface charge of casein micelles decreases, and caseins are released into the serum phase [23]. The dissociation of caseins is temperature-dependent. At low temperatures (4°C), around 40% of caseins dissociated from micelles at pH 5.5, while no virtual dissociation of caseins occurred at 30°C [24, 25]. On the contrary, mineral solubilization is independent of acidification temperature. The extent of mineral solubilization

#### **Figure 1.**

*The structure of casein micelle: Black spheres represent the calcium phosphate nanoclusters that solubilize during the acidification process. Blue coils represent αS and β-caseins; red lines on the outermost part of the surface represent κ-caseins. (Source: Wang and Zhao [4]).*

increases markedly below pH 5.6 and is almost complete at around pH 5.0 [3, 26]. No changes in the hydrodynamic diameter of casein micelles occur during the acidification to pH 5.0 [22], although the charge of κ-casein decreases with acidification, resulting in the collapse of the κ-casein layer and the reduction in the stability of the micelles, as the intra- and inter-chain interactions are insufficient to keep the protein fraction extended in solution [27]. Further decrease to pH around 4.8 results in the aggregation of caseins and the formation of gel structure in unheated milk [2].
