Simultaneous Production of Multi-Functional Peptides in An Enzymatic Membrane Reactor Via Combinational Chromatography

Bioactive peptides derived from bovine casein exert various physiological functions. These peptides ususally included angiotensin converting enzyme inhibitory peptides (ACEIPs), phosphopeptides (CPPs) and antimicrobial peptides (AMPs)^[1]. ACEIPs possess the capacity to lower blood pressure in hypertensive subjects. CPPs, containing the cluster sequence Ser(P)-Ser(P)-Ser(P)-Glu-Glu, can stabilize calcium and phosphate ions, thus enhancing calcium absorption. AMPs are cationic and typically consisting of 12 to 50 residues. They can slow the growth of bacteria and play an important part in the host defense system of many animal species.

Although numerous techniques have been investigated for the preparation of these peptides, the commercial production has been limited due to the lack of suitable large-scale technologies as well as the high cost of enzyme and raw materials. Enzymatic hydrolysis seems to be the most appropriate method for preparation of peptides in nutritional applications, which could be performed in a conventional batch reactor (CBR) or in an enzymatic membrane reactor (EMR) for continuous production of peptides. Several studies have concluded that EMR could result in higher productivities and more uniform products than CBR. However, EMR has been only applied to the preparation of one kind of specific peptide until now.

On the other hand, as reviewed^[2], various potential bioactive peptides have been derived from casein hydrolysates, indicating that screening diverse bioactive peptides simultaneously from one kind of raw material would be achievable. However, most reports only focused on the preparation of one specific peptide, while simultaneous preparation of various peptides was rarely investigated.

Based on the above analysis and considering of high productivity & low cost, three kinds of bioactive peptides (ACEIPs, AMPs and CPPs) were simultaneously prepared from casein by pancreatic hydrolysis via an enzymatic membrane reactor (EMR) (see Fig. 1a, Integrated System) and combinational chromatography (see Fig. 1a, Simultaneously preparation) in this work. The reaction was performed at 37 ^oC and pH 8.0 for 3h followed by ultrafiltration. Strong cation exchange high-performance liquid chromatography (SCE-HPLC) was used to isolate AMPs and CPPs from retentate fractions ranging from 1kDa to 5kDa. Concurrently, ACEIPs were purified by size exclusion chromatography (SEC) from permeate fractions with molecular weight below 1kDa. Fraction 5 corresponding AMPs from SCE exhibited the highest activity against S.aureus (97.49% in 1.0mg/ml) (Fig. 1b). The ACEIPs in fraction B from SEC had the highest ACE-inhibiting-activity (73.5% in 1.0mg/ml) (Fig. 1c). Following reverse-phase high performance liquid chromatography-electrospray ionization tandem mass spectrometry (RP-HPLC-ESI-MS/MS) analysis, potential ACEIPs and a total of 34 CPPs were identified (18 corresponded to alpha_s1-casein, 2 to alpha_s2-casein, 10 to beta-casein and 4 to kappa-casein) (Fig. 1d). This work provided a novel method to Simultaneously prepare multi-functional peptides from casein hydrolysates via enzymatic membrane reactor and combinational chromatography.

This research was supported by the NSF of China (20976125, 31071509, 51173128, 21206113) and Tianjin (10JCYBJC05100), the Ministry of Science and Technology of China (Nos. 2012YQ090194, 2013AA102204, 2012BAD29B05), the Program for New Century Excellent Talents in Chinese University (NCET-08-0386), and Beiyang Young Scholar Program (2012).

References

1.     Silva, S.V. and F.X. Malcata. (2005). Caseins as source of bioactive peptides. International Dairy Journal, 15(1), 1-15.

2.     Phelan, M., Aherne, A., FitzGerald, R. J., & O'Brien, N. M. (2009). Casein-derived bioactive peptides: Biological effects, industrial uses, safety aspects and regulatory status. International DairyJournal, 19(11), 643-654

Figure 1 Simultaneous preparation of AMPs, ACEIPs and CPPs. a) Schematic diagram of the production of AMPs, ACEIPs and CPPs in an enzymatic membrane reactor via combinational chromatography, b) The antibacterial activity against E.coli and S.aureus strains of fractions 4-10 in cation exchange chromatogram; c) Size exclusion chromatogram of FIII on a SephadexG-15 gel column and ACE-inhibitory activity (%) of each fractions with green bars, d) The percentage of the number of phosphorylated peptides.