*2.3. Particle Size Distribution*

The particle size distributions (PSD) for the untreated and ultrasonicated samples are shown in Figure 3a,b. The untreated protein has a peak value of 49.1% in number fraction at 142 nm with a relatively narrow span of 122 nm to 164 nm (42 nm), which indicates that the native protein aggregate was monodispersed in the aqueous systems [5].

The effect of ultrasonic time (5, 10, 15, 20 and 30 min at 20 W/mL) on the PSD pattern of purified protein is shown in Figure 3a. With ultrasonication for 5 min, the peak particle diameter did not change obviously, but the diameter span increased and the minimum particle diameter decreased. With increased ultrasonic time, the peak particle size and diameter span increased. The maximum peak particle size (255 nm) and diameter span (620.7 nm) were obtained by increasing ultrasonic duration to 30 min. The influence of increasing ultrasonic intensity (10 W/mL to 50 W/mL for 20 min) on PPO is shown in Figure 3b. With the intensity of 10 and 20 W/mL, the peak particle diameter did not obviously change, but the diameter span increased. When the ultrasonic intensity increased to 30 W/mL, the particles started aggregation to attain higher particle diameter. The maximum peak diameter (342 nm) and diameter span (863.7 nm) was obtained after ultrasonic processing at 50 W/mL for 20 min.

In general, after ultrasonic processing, the PSD patterns of protein exhibited remarkable changes with wider spans of larger and smaller particle diameters, suggesting the polydispersion and aggregation of protein particles [17]. The native protein aggregate was monodispersed in the aqueous systems because of hydrogen bonding, hydrophobic interaction and electrostatic interaction. A previous study found that ultrasonic processing caused the cleavage of agglomerates, hydrophobic interaction, and van der Waal's forces among protein molecules, thereby inducing the structural changes in enzyme protein [18]. In this case, the particle sizes were decreased because of the shear forces due to cavitation [18,19]. However, as the ultrasonic processing duration and intensity increased, some particles started aggregation. Similarly, Gulseren et al. [20] found that ultrasonic processing at a high duration of 40 min could cause the formation of large aggregates in bovine serum albumin protein. This aggregation might be due to the electrostatic and hydrophobic non-covalent interactions among protein particles [20]. The surface hydrophobicity decreased after prolonged sonication on reconstituted whey protein concentrate, which is a sign of protein aggregation [21]. Under the low-intensity or short-term ultrasonic processing, the enzyme exhibited dissociation because of the forces caused by cavitation. However, the aggregation increased with the increased intensity and time due to noncovalent interactions.

**Figure 3.** Particle size distributions of native and ultrasonic-processed PPO at 20 W/mL for 5, 10, 15, 20 and 30 min (**a**); processed for 20 min at 10, 20, 30, 40 and 50 W/mL (**b**).
