## 2 Advancements in Ultrafiltration for Protein and Peptide Separation
### Integration of UF with Other Processes  
In general, UF is easier to conduct with highly diluted hydrolysate (equal to or lower than 1% \(w/v\)) and low VRF. However, this will lead to an uneconomical process at the industrial scale due not only to productivity reduction, but also to a wider membrane area requirement. Several techniques of UF integration are also plausible to tackle this problem and explicated as follows:  
A study showed the effect of two-step separations of fish protein hydrolysate (FPH) with pressure-driven tangential flow UF and following NF under high VRF value [164]. Solution of FPH1 (Prolastin, main MW 0.3\(-\)1 kDa) and FPH2 (MariRep C, main MW 1-4 kDa) were employed until reaching VRF of 6.0 and 8.3 in the NF step, respectively. They found out that UF was more impactful to fractionate less hydrolyzed FPH (FPH2) and resulted in higher permeate flux compared to NF, but high VRF cannot provide pure fraction of peptides as UF MWCO 4 kDa only produced permeate enriched with FPH below pivot points 0.6-0.75 kDa, and vice versa for retenate side [164]. The deficiency of UF-NF arrangement was also investigated during treatment of dairy effluent, even while shear-induced VSEP and RDM [165]. In part of NF, the absence of casein micelles that was removed beforehand in UF step accentuated the calcium ions to agglomerate the remaining proteins and decline the permeate flux at high VRF. Thus, neither improvement of permeate flux nor energy consumption can be picked up from this configuration. Further observation elucidates three configurations of: (1) UF-NF, (2) diafiltration-UF-NF, and (3) UF-diafiltration-NF for tuna dark muscle hydrolysates [166]. They concluded that configuration 1 was inefficient to fractionate this kind of protein hydrolysate, as NF retenate contained more than 80% of total peptide. Meanwhile, in configuration 2 and 3 NF retenate, the total protein was under 60%. Ultimately, although complete rejection of peptides above 4 kDa was performed by all variation, the permeate flux of configuration 3 was slightly lower owing to high protein content of UF permeate has triggered fouling. Additionally, insertion of diafiltration as a method to bridge the trade-off between yield and purity of the most bioactive peptides (NF permeate enriched with 0.3 kDa peptides) was also mentioned for fractioning peptides from white fish fillet [167]. Other approaches have been examined in order to optimize the selectivity, among others, membrane stacking of UF. Using RC membrane MWCO 30 kDa to separate myoglobin and \(\beta\)-lactoglobulin, 80.25%, 98.31%, and 100% of \(\beta\)-lactoglobulin rejection can be attained by one, two, and three membranes in parallel [168]. This research and other similar studies only examined model protein solution with binary or ternary compound [168; 169; 170]. Thus, the use of internally-staged dead-end UF for tilapia by-product hydrolysate (TBH) as the advancement from the previous research was one of the most representative case owing to the use of complex hydrolysate mixture [171]. Here, a single flat sheet RC UF membrane with MWCO 5 and 10 kDa, and multilayer orientation (top/bottom) of 10/5 and 5/5 kDa were compared in terms of permeate flux, selectivity, and ACE inhibitory activity. From the highest to the lowest permeate flux was developed by combination 10 \(>\) 10/5 \(>\) 5 \(>\) 5/5 kDa. This condition reflected that the first layer where the solution penetrates should be arranged in a way that makes concentration polarization is less intense, as in this case by positioning 10 kDa membrane, which has a more open structure (reducing concentration polarization), on top of the 5 kDa. Interestingly, permeate from 10/5 and 5/5 kDa multilayer combination suggested higher activities of 75.09 and 84.04%, respectively, as opposed to single membrane 5 kDa (71.83%) and 10 kDa (64.32%) since although all of them were enriched with peptides lower than 1.5 kDa, the two first were capable of specifically accumulating more peptides below 0.5 kDa [171].