*3.5. Ultrasonic Processing*

An ultrasonic processor (JY92-2D, Ningbo, China) containing a titanium probe of diameter 0.636 cm was used to sonicate 10 mL of protein in 25 mL of centrifugal tubes, surrounded by ice to maintain the low temperature. All ultrasonic processing was definitely below 30 ◦C. The protein samples were treated at a low frequency of 20 kHz with the pulse duration of 5 s on and 5 s off setting, to investigate the effect of ultrasonic time (10, 15, 20 and 30 min at 20 W/mL) and intensity (10, 20 and 40 W/mL for 30 min) on the protein. The ultrasonic-processed samples were stored at 4 ◦C for further analysis.

#### *3.6. PPO Activity Assay*

The activity of the PPO enzyme was measured by determining the increasing rate of absorbance per minute at 420 nm using an Eppendorf Bio-spectrometer (Eppendorf, BioSpectrometer kinetic, Hamburg, Germany). The protein solution of 0.5 mL was mixed with 0.1 mol/L catechol (1 mL) and 0.1 mol/L Tris-HCl buffer (1 mL), then the absorbance of the resultant mixture was measured at 420 nm [5]. The relative activity (RA) of the PPO enzyme was measured according to the following equation.

$$\text{Relative PPO activity} = \frac{\text{PPO activity of ultrasonic treated } ppo}{\text{PPO activity of native prop}} \times 100\% \tag{1}$$
