# Effect of Whey Protein Changes on Milk Flavor and Sensory Characteristics During Heating
## 3 Results and Discussion  
### Protein Profile and Structural Changes  
3.1.1. Protein Denaturation and Aggregation  
SDS-PAGE is increasingly applied in milk protein studies due to its high resolution and quantitative results [26]. Reducing and non-reducing SDS-PAGE were performed to determine the protein composition, aggregate formation, and protein denaturation of milk after different heat treatments. Figure 1A,B show the electrophoretic profiles of the protein solutions during SDS-PAGE after different heat treatments. No significant changes were evident in the \(\alpha\)-CN and \(\beta\)-CN bands as the treatment temperatures increased. Contrarily, \(\beta\)-LG and \(\alpha\)-LA, the main components in milk whey proteins, are thermally unstable due to their significant number of secondary and tertiary structures [27]. The denaturation of these components started at 65 \({}^{\circ}\)C and mainly occurred when the milk was heated above 80 \({}^{\circ}\)C [14]. When the temperature increased to 75 \({}^{\circ}\)C and 100 \({}^{\circ}\)C, the \(\beta\)-LG and \(\alpha\)-LA bands became lighter and more obvious in the non-reducing state. The non-reducing SDS-PAGE protein bands were less obvious than those in reducing conditions, indicating that heat-induced protein aggregation occurred mainly via covalent bonding. This caused protein aggregates that were difficult to separate [28; 29]. In reducing conditions, the \(\beta\)-LG displayed no significant changes when heated at 60 \({}^{\circ}\)C for 10 min, 60 \({}^{\circ}\)C for 30 min, and 75 \({}^{\circ}\)C for 10 min (Figure 1A). Since \(\beta\)-LG was in a \"molten globule state\" at this time, the conformational change was reversible [30]. Figure 1B shows the electrophoretic profile in non-reducing conditions. The \(\beta\)-LG, \(\alpha\)-LA, and LF bands disappeared gradually as the heating temperature increased to 75 \({}^{\circ}\)C and 100 \({}^{\circ}\)C, which was consistent with the results of previous research [31]. This differed significantly from the results in Figure 1A, possibly because heating caused protein unfolding to expose the thiol groups. This caused protein aggregation and complex formation between \(\alpha\)-LA and \(\beta\)-LG, \(\alpha\)-LA and \(\kappa\)-CN, and \(\beta\)-LG and \(\kappa\)-CN via thiol-disulfide exchange reactions [14; 32]. Furthermore, due to higher heat sensitivity, the \(\beta\)-LG bands were lighter than those of \(\alpha\)-LA [27]. This study primarily examined whey proteins in subsequent experiments due to their sensitivity to heat treatment and tendency to decrease in content at higher temperatures.  
Figure 1: Changes in the milk proteins after different heat treatments. (**A**) The SDS-PAGE profile in reducing conditions. (**B**) The SDS-PAGE profile in non-reducing conditions. (**C**) The denaturation rates of the active proteins (\(\alpha\)-LA, \(\beta\)-LG, and LF). (**D**) The CD analysis of the whey protein solution. \({}^{\mathrm{a-f}}\) According to the Tukey test, different superscripts of the same active protein indicate significant differences (\(p<0.05\)).