(585p) 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.