# Effect of Whey Protein Changes on Milk Flavor and Sensory Characteristics During Heating
## 3 Results and Discussion
### Protein Profile and Structural Changes
#### 3.1.3 Secondary Structures of the Whey Proteins  
\(\alpha\)-LA and \(\beta\)-LG are globular proteins that naturally fold into unique, compact, highly ordered structures. Regular conformational regions, known as \(\alpha\)-helix and \(\beta\)-folds, are interconnected to form a 3D structure [36; 37]. Proteins undergo structural alterations during processes such as ligand binding. Figure 1D shows the changes in the secondary whey protein structures after different heat treatments. The CD band of the protein in the far ultraviolet region (190-260 nm) was mainly derived from the amides of the protein backbone and was sensitive to their conformations [18].  
The \(\alpha\)-helix displayed a positive peak at 190 nm and a negative peak at 208 nm in the CD spectrum due to \(\pi_{\text{nb}}\)\(\pi^{\text{+}}\) transition exciton splitting, while another negative peak was evident at 220 nm due to \(\pi\pi^{\text{+}}\) transition [38]. The \(\beta\)-sheet exhibited a positive peak near 195 nm and a negative peak near 215 nm, while the \(\beta\)-turn displayed a maximum peak at 200 nm and a minimum peak at 220 nm. Furthermore, the proteins without dominant secondary structures (random coil) presented a negative peak near 200 nm [38; 39]. Thermal denaturation of milk typically involves protein molecule unfolding and the irreversible aggregation of unfolded molecules [40]. As shown in Figure 1D, the raw sample displayed a \(\beta\)-sheet structure. Compared with the raw samples, the maximum and minimum values of the CD spectra at 195 nm and 215 nm of 6010 and 6030 blue-shifted to 190 nm and 208 nm, respectively, while the negative ellipticity gradually increased between 208 and 230 nm. The peak shifts indicated protein \(\beta\)-sheet loss, while the \(\alpha\)-helix and random coil content increased, indicating that the whey protein changed from an ordered to a disordered structure after heat treatment [41]. The CD spectra of the whey proteins were similar in the milk heated at 75 \({}^{\circ}\)C and 100 \({}^{\circ}\)C, with a significant negative peak near 200 nm, indicating an increase in the random coil content. The CD spectra results indicated that the different heat treatments altered the secondary structures of the milk whey proteins. Enhanced hydration and partial tertiary structure loss were evident at 50 \({}^{\circ}\)C. The mobility of certain polypeptide chain sections increased at a pasteurization temperature between 65 \({}^{\circ}\)C and 71 \({}^{\circ}\)C, revealing secondary structures such as \(\alpha\)-helix and \(\beta\)-sheet. Temperatures between 70 \({}^{\circ}\)C to 80 \({}^{\circ}\)C caused disulfide bond breakage, tertiary structure loss, and partial secondary structure loss. Disulfide bond breakage and secondary structure loss were evident between 80 \({}^{\circ}\)C and 90 \({}^{\circ}\)C, while temperatures between 90 \({}^{\circ}\)C and 100 \({}^{\circ}\)C caused intermolecular disulfide bond formation and hydrophobic interactions. Protein coagulation, lysine formation, and Maillard reactions were apparent between 100 \({}^{\circ}\)C and 105 \({}^{\circ}\)C [42].  
Table 1 shows the percentages of the whey protein structural changes after different heat treatments, with the \(\alpha\)-helix in the raw sample at 15.5%, the \(\beta\)-sheet at 34.6%, andthe \(\beta\)-turn at 19.6%. When heated at 60 \({}^{\circ}\)C, the proportion of \(\alpha\)-helix and random coil increased, while the \(\beta\)-sheet decreased. Furthermore, a higher temperature and extended time increased the random coil percentage to 31.8% in 7510 and 32.0% in 10010, and the \(\beta\)-turn increased, which was consistent with the results of previous research [18]. In summary, higher treatment temperatures altered the secondary structure and interaction forces of the whey proteins, which affected the milk flavor.