The SFPU experiments were done in an ultrasonic bath reactor (internal dimensions: 362 mm × 294 mm × 502 mm, Shangjia Biotechnology Co., Wuxi, Jiangsu, China) equipped with five different frequency plates (Fig. 1a) and the maximum output acoustic power of each plate is 600 W. The bath ultrasound generators provided direct contact with the surface of the solution with a bath ultrasound (upper plate) as a power source for acoustic cavitation. Another bath ultrasound (bottom plate) was placed at the bottom of the reactor. The SFPU generator provided with function modes of sweeping frequency operation and pulsed operation. The sweeping frequency operation (fi ± δ kHz) refers to the sweeping frequency Cathepsin G Inhibitor of increasing period from fi − δ to fi + δ kHz and decreasing period from fi + δ to fi − δ kHz with the same linear speed in the form of an isosceles triangle ( Fig. 1b), and the pulsed operation indicates that ultrasound is generated in a pulsed mode with an on-time and an off-time cycle. A 500 mL CGM suspension (net protein content of 50 g L−1) were sealed in a high pressure resistance bag and pretreated with SFPU. The ultrasonic pretreatment parameters were obtained from our previous studies as follows: combination of ultrasonic sweeping frequency 28 ± 2 kHz (upper plate) and 68 ± 2 kHz (bottom plate), temperature 30 ± 2 °C, pulsed on-time 10 s and off-time 3 s, cycle time of the sweeping frequencies of 500 ms, duration of 40 min and power density 80 W/L.