## 2 Advancements in Ultrafiltration for Protein and Peptide Separation
### Ultrasonic-Assisted UF  
Ultrasound (US) has been used to aid broad spectra of separation process like feed pretreatment [118; 119; 120; 121], enzymatic hydrolysis [55], extraction, and centrifugation [122; 123], including membrane UF [82; 124; 125; 126], with special attention toward reduction of fouling and concentration polarization. By transferring considerable mechanical power through small mechanical movements from liquid (high intensity) into a gaseous medium, US can work to disrupt the structure of the cake layer and concentration polarization covering the surface of membrane. US transition and propagation are comprised of two foremost phenomena categories, which are (1) cavitation (liquid jets), radiation pressure, and acoustic streaming and (2) physicochemical transformation (dispersion, coagulation, and liquid property changes) [127]. To illustrate the performance of US, pretreatment of egg white protein (EWP) by US and subsequent UF was conveyed to endow more antioxidant features (measured by DPPH and ABTS methods) of low molecular weight peptides (<1 kDa as much as 11.19 \(\pm\) 0.53%, and 1-10 kDa for 28.80 \(\pm\) 0.07%) [55]. Likewise, whey protein concentrate pretreated shortly (below 5 min at 20 kHz) by US showed reduction in viscosity owing to smaller aggregates [128], and advanced heat stability [129]. In treating cheese whey wastewater with UF, ultrasonication was used as a method of reducing chemicals for membrane cleaning [82]. It is also shown in another work that the treatment with ultrasonication (20 kHz, power 300 W) was more effective for irreversible fouling, which gave a 17.23% increase in cleaning efficiency of permanently hydrophilic PES as opposed to 5.47% in PES (30 kDa). In the end, profitability analysis provided the data that only small proportion of additional cost was needed for US in comparison with NaOH treatment [82]. Some research studies have shown that better cleaning performances can be obtained from lower frequencies of US and higher power intensity since high intensity implies in the detriment of membrane material [130] and protein denaturation [131]. However, the collapse of larger bubbles generated from acoustic cavitation at low US frequencies resulted in microjetting and turbulence localization, insocuch as the less intense of cavitation collapse from smaller bubbles at higher frequencies [132]. Indeed, low US frequency has better cleaning efficiency and flux performance, but a slight protein denaturation can still inflict in the form of self-aggregation with free casein micelles in the first 30 min of ultrasonication, even at low frequency (20 kHz) with power 20 and 41 W. After all, the viscosity of milk and casein micelles were not transformed by acoustic cavitation, and the size of fat globules decreased with ultrasonication at lower power [133]. Submerged module in US bath also had higher efficiency than placing US in the cleaning solution as a novel way of US membrane cleaning if protein is attached cohesively with membrane surface by cationic bonding due to calcium-protein interaction. Otherwise, it would not expose a different inclination in cleaning efficiency [125]. The cleaning efficiency is more related with the conditions of fouling solution. For instance, despite  
Figure 4: Scaling and fouling in EDUF during protein separation. (**a**) Mechanism of induction of major scaling agents: Ca(OH)\({}_{2}\), CaCO\({}_{3}\), and Mg(OH)\({}_{2}\). (**b**) Plausible transport of casein, \(\alpha\)-lactalbumin, and \(\beta\)-lactoglobulin in pH 6.8–5.0. Membrane with MWCO larger than 10 kDa is not recommended for this procedure since whey proteins and individual caseins may penetrate through the CEM and cause organic fouling. (**c**) Conventional five-compartments with represented scaling on CEM. (**d**) New 6-compartments without observed scaling on CEM. Panels (**a**) and (**b**) are reproduced from [117] with permission, © 2021 Elsevier Ltd. Panels (**c**) and (**d**) are reprinted from [81] with permission, © 2021 Royal Society of Chemistry).