ELSEVTER

Desalination

149 (2002) 369-374

The development of electro-membrane filtration for the isolation of bioactive peptides: the effect of membrane selection and operating parameters on the transport rate
G. Bargeman*, G.-H. Koopsb, J. Houwing, I. Breebaartb, H.C. van der Horsta, M. Wesslingb
INIZO Food Research, Tel. ~31 (318) 659565; Fax +3/ (318) 650400; University of Twente, Faculty of Chemical emails: g.h.koops@ct.utwente.nl, Received PO. Box 20, 6710 BA Ede, the Netherlands emails: bargeman@nizo.nl, j.houwing@tnw.tude[ft.nl, horst@nizo.nl Technology, P 0. Box 217, 7500 AE Enschede, The Netherlands i. breebaart@ct.utwente.nl, m. wessling@ct.utwente.nl

1 February 2002; accepted 11 March 2002

Abstract The ability to produce functional food ingredients from natural sources becomes increasingly attractive to the food industry. Antimicrobial (bioactive) ingredients, like peptides and proteins, can be isolated from hydrolysates with membrane filtration and/or chromatography. Electra-membrane filtration (EMF) is an alternative for the isolation of these usually strongly charged components. It is believed to be more selective than membrane filtration and less costly than chromatography. The isolation of bioactive peptides from a hydrolysate of a,-casein, a protein originating from milk, was studied as a model separation for the development of EMF. This separation can be used as an example application for the isolation of other charged components from complex feedstocks in sseveral industries. After 4 h EMF the product consisted for 100% of proven or anticipated charged bioactive components. Diffusion and convection were negligible in relation to electrophoretic transport, since only charged components were recovered in the permeate product. The most important peptide (26% on total protein, starting from 7.5% in the feed) was a,caseinfi 183-207), a very potent peptide against Gram positive and Gram negative microorganisms. The transport rate of as2-caseinf( 183-207) was reduced strongly when a polysulphone membrane with a molecular weight cut-off below 20 kDa was used. The amount of a.,-caseinf(183-207) transported increased practically linearly with the concentration and the applied potential d&reference. The use of desalinated feeds to further increase the electrical field strength in the feed compartment resulted in higher transport rates, but this increase was lower than expected probably due to the lower electrophoretic mobility. An average transport rate of 2.5 and 4 g/m?.h at maximum was achieved during 4 h EMF using GR60PP (25 kDa) and GR41PP (100 kDa) membranes respectively. Keywords: Electrophoresis; *Corresponding author. Congress on Membranes and Membrane Processes (ICOM), Toulouse, France, Nutraceuticals; Ultrafiltration; Food; Peptides; Bioactive

Presented at the International July 7-12, 2002.

00 11-9 164/02/$- See front matter 0 2002 Elsevier Science B.V. All rights reserved
PII:SOOlL9164(02)00824-X

370

G. Bargeman

et al. /Desalination

149 (2002)

369-374

1. Introduction
In the food industry, there is a clear trend towards the production of so-called functional foods and nutraceuticals. In these products biologically active ingredients from natural sources, e.g. dairy proteins or protein fragments are believed to be responsible for the health- and wellbeing-promoting effects [ 1,2]. Anti-microbial peptides or proteins are usually strongly charged. The presence of this charge is thought to be essential for their bioactivity [3]. For the production of functional foods, addition of a mixture of charged bioactive components is usually acceptable and the isolation of one very pure peptide or protein is therefore not required. Currently membrane filtration and/or chromatography are used for the isolation of these components. Electra-membrane filtration (EMF) seems to be a promising alternative. EMF combines conventional membrane filtration with electrophoresis (Fig. 1). As compared to pressure-driven membrane filtration an increase in selectivity for the isolation of charged components is anticipated. Furthermore it is probably less costly than chromatography. Only a few publications deal with the isolation of valuable charged components from industrially available feeds. The isolation of antibodies from ascitic fluids on an analytical scale and of amino
Electrode Feed Permeate Electrode

acids and bioactive components from hydrolysates are described by Lim et al. [4] and Bargeman et al. [5] respectively. The feasibility of EMF for the isolation of charged amino acids and bioactive peptides from protein hydrolysates was proven in scouting experiments on lab-scale. Lysine was successfully isolated from a casein hydrolysate containing other neutral amino acids of similar size. A product purity of 96% was obtained [5]. Furthermore a mixture of positively charged peptides with proven and anticipated bioactivity was isolated from a lactoferrin hydrolysate. In this mixture the main component was lactoferricin a highly potent anti-microbial peptide [5]. In this publication the isolation of positively charged peptides, and especially the bioactive peptide a,-casein fragmentfl183-207), from an q,-casein (a,,-CN) hydrolysate with EMF is described. With conventional UF diafiltration using GR60PP the isolation of bioactive peptides from as,-CN hydrolysate could not be achieved [6]. The effect of the membrane molecular weight cut-off (MWCO) and operating parameters on the transport rate of a,-CNJ9183-207) during EMF is reported. This separation can be used as an example application for the isolation of other valuable charged components from complex feedstocks in e.g. the food, pharmaceutical, fine chemical and fermentation industries. 2. Materials and methods

2. I. Production of hydrolysates and desalinated

hydrolysates
4-o

0--, m

Cation Anion 0

Cationic peptide Uncharged peptide

Protein

Fig. 1. EMF principle (CEX: cation exchange membrane; AEX: aniion exchange membrane; UF: ultrafiltration membrane).

A solution containing approximately 25% as?CN was prepared from sodium caseinate (DMV International Veghel, The Netherlands) as described by Bargeman et al. [6]. The as,-CN solution was hydrolysed using 6% (m/m of substrate) porcine pepsin A (EC 3.4.23.1, 439 units/mg solid) [6]. Subsequent freeze-drying of the hydrolysate according to the procedure described by Bargeman et al. [6] produced three different batches of hydrolysate powder (respectively hydrolysate A,

G Bargeman et al. /Desalination

149 (2002) 369-374

371

B and C), with only marginally different peptide concentrations. Desalinated hydrolysate batches D and E were produced from hydrolysate batch B through diafiltration using NF40 (DOW FilmTech) and hydrolysate batch C through diafiltration using NF45 (DOW Filmtech), respectively. Feedstocks for EMF experiments were produced by dissolving the freeze-dried desalinated or nondesalinated hydrolysate powder to the required concentration. The solutions of c,=2.4 g/l hydrolysate A and c,=2.0 g/l hydrolysate D had a similar peptide concentration, based on the absorbance at 280 nm (A&. 3. Experimental Batch-wise EMF experiments were carried out in laboratory EMF equipment [5,6] with a membrane area of either 0.008 m2 (rig 1) or 0.010 m* (rig 2). The cell pair thickness of rig 1 was20mm,5mm,15mmand5mmfortheanode, the feed, the permeate and the cathode compartments, respectively. For rig 2 the cell thickness of both the feed and the permeate compartment was 2.1 mm. The feedstock, as well as the permeate were recycled over the module without bleeding either of the two. At the start of each run the permeate and electrode compartment contained a 20 or 50 mM Na,SO, (pa, Merck, Darmstadt, Germany) solution in demineralized RO water
Table 1 Conditions of EMF experiments Experiment number 1 2 3 4 5 6 7 8 9 10 AV, V 40 60 40 40 40 28.8 28.8 28.8 28.8 28.8 Initial c,, cp, mM Na2S04 20 20 20 50 20 20 20 20 20 20 Membrane GR60PP GR60PP GR60PP GR6OPP GR60PP GR60PP GR81PP GR4OPP GR61PP GRSlPP

(rig 1) or in deionized water (18 mS/cm MilliQ, rig 2). During the experiments the pH of the feed solution was maintained at pH 8.Oti.5 by titration with 0.2 M NaOH (Merck, Darmstadt, Germany). The pH of the permeate solution was maintained at pH 4.020.5 by addition of 0.2 M HCl (pa, BDH lab supplies, Poole, UK) during the experiments in rig 1 and between pH 4-6 by addition of a 0.2 M H,SO, solution during the experiments in rig 2. CMX and AMX (Tokoyama Soda, Japan) ion-exchange membranes were used to prevent direct contact between feed and product with the electrodes (Fig. I). For the isolation of bioactive peptides from an as?-CN hydrolysate ultra.tYtration polysulphone membranes (DOW FilmTec, DSS Denmark) with MWCO between 10 kDa and 100 kDa (Table 1) were used to study the effect of MWCO on the transport rate. The transport of components through the UF membrane can be described by Maxwell-Stefan, e.g. [7] or extended Nemst-Planck models [8]. To get a quick impression of the parameters that influence the transport rate the extended NemstPlanck equation is very suitable:

The molar transport rate of the components through the membrane, Ji [see Eq. (l)] is due to

Mwco, kDa 25 25 25 25 25 25 10 100 20 50

Feed type A A A E E F F F F F

c/; g hydrol.fL 2.4 2.4 2.4 0.8 2.0 2.5 2.5 2.5 2.5 2.5

EMF rig 1 1 1 1 1 2 2 2 2 2

372

G Bargeman

et al. /Desalination

149 (2002)

369-374

convection, diffusion and electrophoretic transport, respectively. To obtain a high transport rate all contributions have to be maximized. However, since the isolation of especially charged components is targeted, the contribution of the electrophoretic transport as a consequence of the electrical field strength in the feed compartment Ef has to be maximized in relation to convection (and diffusion). EMF was therefore carried out at low pressures (below 0.1 bar). The experiments were carried out at approximately 20C. During the experiments the concentration, feedstock conductivity and the applied potential difference were varied to validate the effect of these variables on the transport rate. A summary of the experimental conditions apphed is presented in Table 1. 4. Results and discussion The EMF permeate (product) only contained positively charged peptides (besides small positively and negatively charged salt ions). Negatively charged or neutral protein fragments were not recovered in the permeate, indicating that the electrical field strength was the main driving force as intended [6]. Most out of the approximately ten components isolated are proven antimicrobial peptides. The remaining components in the product are believed to show antimicrobial activity on the basis of their positive charge [6]. The main component in the product was the charged ~,~~-CNfil83-207), highly potent against Gram positive and Gram negative microorganisms. The EMF product contained 26% (based on the RP-HPLC peak area) at maximum of this bioactive peptide, starting from 7.5% in the feedstock. Consequently, EMF is capable to produce from a,,,-CN hydrolysate a valuable product consisting for probably 100% of bioactive components and for 26% of the proven bioactive a,-CN fll83207) peptide. The transport rate of as,-CN f( 183-207) depends on the MWCO of the UF membrane used

30

20

40 MWCO

60

80

100

(kDa)

Fig. 2. The effect of MWCO on the average transport rate of or,s~-CNf(183-207) after 4 h batch-wise EMF (rig 2).

(Fig. 2). For a MWCO of 10 kDa (approximately 3 times the molecular weight of a,-CN A183207), the transport rate was more than a factor 3 lower than for the membranes with higher MWCO. This is ascribed to the higher friction of the peptide in the membrane pores for the low MWCO membrane, resulting in retardation of the peptide transport. For MWCO in excess of 20 kDa (approximately 6 times the molecular weight of as,-CN f( 183-207) this friction plays a less important role. So far average transport rates of 2.5 g/m*.h and 4 g/m*.h at maximum were obtained during 4 h EMF operation for GR60PP (25 kDa) and GR40PP (100 kDa), respectively (Fig. 2). A reduction of the hydrolysate concentration from 2.0 g/l to 0.8 g/l resulted in a practically linear reduction of the amount of cc,-CN j(183-207) transported to the permeate (M.A.X(J.At) = 42 vs. 18 mg) after 4 h EMF. A further increase of the transport rate therefore seems to be feasible by increasing the hydrolysate concentration. The marginal deviation from the linear relation between the concentration and the amount transported is explained as follows. During EMF of the feedstock with 0.8 g/l hydrolysate, Efwas slightly higher than during EMF with the feedstock containing 2.0 g/l hydrolysate, as a consequence of the marginally lower initial feed conductivity

G Bargeman et al. /Desalination

149 (2002) 369-374

373

and the higher conductivity of the permeate and electrode solutions. Consequently the amount of aT2-CNfi 183-207) transported at low hydrolysate concentration was somewhat higher than expected on the basis of the feed concentration alone [see Eq. (1 )]. Nevertheless the higher electrical field strength in the feed compartment was used less efficiently as shown by the effect of the cumulative field strength on the amount transported (Fig. 3) to the permeate. This may be caused by a relatively high adsorption of a,-CN f( 183-207) at low concentrations. In general, the use of non-desalinated feed resulted in slightly lower transport rates [6]. This is due to the lower electrical field strength in the feed compartment at similar overall potential difference as a consequence of the higher feed conductivity. However, the difference in the amount of a,,,-CNf(l83-207) transported during the same period of time was smaller than anticipated, since the electrical field strength in the feed compartment was used more efficiently than for desalinated feed (Fig. 3, non-desalinated feed 2.4 g/l with desalinated feed with c,=

2.0 g/l can be compared directly, since peptide con-centrations are the same). This is probably due to the very low electrophoretic mobility for the desalinated feed at the start of the experiment, as a consequence of the low counter-ion concentrations resulting in retardation of the transport. For EMF at overall potential differences of 40 and 60 V the relation between the cumulative electrical field strength in the feed compartment and the amount of a,-CNfll83-207) recovered in the permeate is the same (Fig. 3). The amount of 0,2-CNJT183-207) transported after 3 h EMF at 60 V was a factor 1.4 higher than for operation at 40 V (M.A.Z (J.At) = 26 vs. 18 mg), practically in line with the ratio of the electrical field strengths in the feed compartment for both situations. 5. Conclusions The feasibility of EMF for the isolation of bioactive peptides from an as,-CN hydrolysate is proven. EMF of this hydrolysate can produce a permeate consisting for 26% of the potent bioactive peptide a$,-CNfi 183-207), starting from 7.5% in the feed. All the other components in the product are also proven or anticipated antimicrobial peptides. For the isolation of as,-CN fil83-207), membranes with a MWCO in excess of 10 kDa (3 times the molecular weight of the peptide) should be used to avoid a strong reduction of the transport rate of the target peptide, probably as a result of increased friction in the pores of the membrane. For a desalinated hydrolysate solution during 4 h EMF operation average transport rates of 2.5 g/m2.h and 4 g/m2.h at maximum are obtained for GR60PP (25 kDa) and GR40PP (I 00 kDa), respectively. Reduction of the hydrolysate concentration results in a practically linear reduction of the amount of as,-CN JT183-207) recovered in the permeate product. The use of a feed with higher conductivity, a non-desalinated hydrolysate solution, results in a slight reduction of the amount of as,-CNfl183-207) transported to the permeate. However, this reduction is smaller than anticipated on the basis of the difference in

0 Non-desalinated feed, c=2.4g/l. 40V Nondesalinated feed, c=2.4g/l, 60V 0 Non-desalinated feed, c=2.4g/l, 40V A Desalinated feed, c=2.Og/l, 40V *Desalinated feed, c=O.6g/l. 40V

0 0 5000 10000 15000 20000 25000

CEf A.t (kV s mu)

Fig. 3. Amount of a,,-CNf(l83-207) peptide recovered in the permeate as a function of the cumulatively applied driving force (rig 1).

374

G. Bargeman et al. /Desalination

149 (2002) 369-374

the electrical field strength in the feed compartment. The low conductivity of the desalinated feed at the start of the experiments probably leads to retardation of the transport due to a reduced availability of counter-ions. An increase of the overall potential difference from 40 to 60 V leads to a practically linear increase in the amount of CX~-CNA 183-207) transported to the permeate. A further increase of the achieved transport rates therefore seems possible by increasing the hydrolysate concentration and the potential difference. The effect of the feedstock conductivity on the transport rate can be improved by optimizing the product of the electrical field strength in the feed compartment and the electrophoretic mobility. The effect of process parameters and the MWCO of the membrane on the separation selectivity and energy consumption should be further investigated. Acknowledgements The authors highly appreciate the financial support of the Dutch program on Ecology, Economy and Technology (E.E.T.). We furthermore like to thank I. Recio, M. Dohmen, R. Hollernan, C. Slangen, D. Stoffels, N. Janssen and J. Leenders. Symbols A ; F J k M u x 2 Membrane area, m* Concentration, mol/l or g/l Diffusion coefficient, m*/s Faradays constant (9.6x104), C/m01 Transport rate, mol/ m*.h or g/m*.h Convective coupling coefficient Moleweight, g/mol Electrophoretic mobility, m.mol/N.s Distance, m Valance

Greek

At AV

Time interval, s Applied overall potential difference,

Subscripts f e i P Feed Electrode solution or compartment Component i Permeate solution or compartment

References Ill
H. Meisel and E. Schlimme, Bioactive peptides derived from milk proteins: ingredients for functional foods?, Kieler Milchwissenschaftliche Forschungsberichte, 48 (1996) 343-357. I. Gill, R. Lopez-Fandina, X. Jorba and E.N. Vulfson, Biologically active peptides and enzymatic approaches to their production, Enz. Microb. Technol., 18 (1996) 162-183. I. Recio and S. Visser, Identification of two distinct antibacterial domains within the sequence of bovine as,-casein, BBA, 1428 (1999) 314-326. S. Lim, HP Manusu,A.A. Gooley, K.L. Williams and D.B. Rylatt, Purification of monoclonal antibodies from ascitic fluid using preparative electrophoresis, J. Chromatogr. A., 827 (1998) 329-335. Ci Bargeman, M. Dohmen-Speelmans, I. Recio, M. Timmer and H.C. van der Horst, Selective isolation of high-value dairy components by electro-membrane filtration, Lait, 80 (2000) 175-186. Ci Bargeman, J. Houwing, I. Recio, G-H. Koops and H.C. van der Horst, Electra-membrane filtration for the selective isolation of bioactive peptides from an alp,-casein hydrolysate, submitted to Biotechn. Bioeng (2001). J. Straatsma, G Bargeman, H.C. van der Horst and J.A. Wesslingh, Can nanofiltration be fully predicted by a model? J. Membr. Sci., 198 (2002) 273-284. R. Schlbgl, Membrane permeation in systems far from equilibrium, Ber. Bunsenges. Phys. Chem., 70 (1966) 400.

121

I31

t41

PI

[61

[71

C81