3.4.1. Flexibility and Turbidity

The flexibility of a protein can be understood as the ability of its structure to change when the external environment of the protein changes, reflecting the sensitivity of the protein structure to environmental changes [41]. It has received increasing attention because of its key role in determining the functional properties of proteins, especially the interfacial properties. Studies have shown that bovine serum albumin with higher flexibility is more likely to form a better viscoelastic protein film at the interface, thereby showing better emulsifying properties [42]. In this study, it was found that when the heat treatment time was certain, QPI flexibility increased and then decreased with the increase of temperature, reaching a maximum value of 0.42 at 90 ◦C, 30 min, which was significantly higher than that of untreated 27.27%. In the range of 60–90 ◦C, the flexibility of QPI gradually increased with the prolongation of heat treatment time. Previous studies have shown that rigid heatresistant proteins reach the flexibility of unstable proteins at higher temperatures [43]. This is probably due to the fact that moderate heat treatment will destroy the covalent or noncovalent forces that maintain the rigid structure of the protein, such as van der Waals forces, hydrogen bonds, electrostatic interactions, disulfide bonds, hydrophobic interactions, etc., thereby increasing the flexibility of the protein [44]. When the temperature was 100 ◦C and 121 ◦C, QPI flexibility then gradually decreased with time and fell to the lowest value of 0.37 at 121 ◦C, 30 min, which was still higher than the untreated 12.12% (Figure 3A). This is probably due to the excessive heating temperature that severely denatures the protein, forming a large number of insoluble agglomerates and decreasing solubility, which in turn leads to reduced flexibility [45].

**Figure 3.** Effect of different heat treatment conditions on flexibility (**A**) and turbidity (**B**) of QPI. Different letters (a–c) indicate significant differences at same temperature (*p* < 0.05).

The measurement of turbidity can directly reflect the dispersion state, aggregation state and particle size of protein particles in the solution [46]. In this study, it was found that the turbidity of QPI showed a gradual increase with the increase of time and temperature. At lower temperatures (60 ◦C and 70 ◦C), the effect of heat treatment time on QPI turbidity was not significant, while at higher temperatures the effect was significant, and QPI turbidity increased continuously with time, reaching a maximum value of 0.94 at 121 ◦C and 30 min, which was 4.53 times higher than that of untreated (Figure 3B). This is consistent with the result that heat treatment increases the turbidity of whey protein [47]. Previous studies have shown that the increase in turbidity is related to the formation of protein molecular aggregates [48]. In addition, higher aggregation rates and higher turbidity increases were also observed for longer microwave heating times for grass carp sarcoplasmic and myogenic fiber proteins [49]. Therefore, we speculate that the increase in turbidity may be due to the unfolding of QPI molecules, when treated at higher temperature and for a long time, the intramolecular and intermolecular interaction forces of proteins are enhanced, resulting in substantial aggregation of proteins [50].
