electrochemical modification of dairy-based functional

ORIGINAL PAPER

Electrochemical modification of dairy-based functionalpeptides by means of cross-flow electromembrane filtration

Aline Holder & Lutz Großmann & Jochen Weiss &Jörg Hinrichs

Received: 24 April 2014 /Revised: 27 June 2014 /Accepted: 18 August 2014 /Published online: 11 September 2014# INRA and Springer-Verlag France 2014

Abstract The stability of functional peptides against electrochemical modificationduring their selective fractionation via cross-flow electro membrane filtration is aprerequisite for their successful application in food products to ensure the bio-function-ality, emulsification, foam formation, or stabilizing properties. This study investigatedthe impact of electrical fields applied to cross-flow membrane filtration on electrolysisand the associated electrochemical modification of functional dairy-based peptides tocalculate the electrical load of target analytes during the process. Firstly, electrolysis andthe hypochlorite-induced oxidation of the model peptide β-CN f(108–113) and lactoseat different hypochlorite concentrations were studied under well-controlled laboratoryconditions, followed by the electrochemical modification of functional peptides duringcross-flow electro membrane filtration. At laboratory conditions, a chemical modifica-tion of the model peptide β-CN f(108–113) below a value of 3 mol.L−1 hypochloritewas not observed, while lactose was modified at minimal hypochlorite concentrationsand, thus, acted as a protection group for peptides. The electrochemical modification offunctional peptides was only observed for voltages ≥10 V in cross-flow electro mem-brane filtration experiments. In addition, results indicate that a further increase inelectrical voltage (>5 V) does not result in a significant improvement of peptideelectrophoresis, and thus, there is no higher fractionation of functional peptides. Theseexperiments show that under optimal conditions of filtration with a superimposedelectrical field, no chemical modification of functional peptides takes place that indicatesan advantage compared to conventional filtration processes by significantly increasingthe fractionation efficiency at voltages between 5 and 7 V.

Dairy Sci. & Technol. (2015) 95:47–62DOI 10.1007/s13594-014-0187-0

This paper is part of the special issue dedicated to the 2nd International Symposium on Minerals & DairyProducts (MADP2014) held on 26-28th February in Auckland, New Zealand.

A. Holder (*) : L. Großmann : J. HinrichsDepartment of Dairy Science and Technology, Institute of Food Science and Biotechnology, University ofHohenheim, Garbenstr. 21, 70599 Stuttgart, Germanye-mail: [email protected]

J. WeissDepartment of Food Physics and Meat Science, Institute of Food Science and Biotechnology, Universityof Hohenheim, Garbenstr. 21, 70599 Stuttgart, Germany

Keywords Peptidemodification .Hypochlorite . Cross-flowelectromembrane filtration. HPLC .Mass spectrometry

1 Introduction

Electrical fields have been reported to have strong fractionation efficiency on proteins,peptides, and amino acids (Daufin et al. 1995; Hofmann and Posten 2003; Huotari et al.1999; Käppler and Posten 2007; Lentsch et al. 1993). Major advantages superimposingan electrical field during cross-flow ultrafiltration are a selective fractionation based onthe physicochemical properties of molecules (size, charge, and adsorption characteris-tics). Electrophoresis principle applied in electrodialysis and pressure-driven filtrationcell has been used for nutraceutical applications, such as the isolation of bioactivepeptides and amino acids (Bargeman et al. 2002; Daufin et al. 1995; Poulin et al. 2006),for the isolation of lactoferrin (Brisson et al. 2007; Ndiaye et al. 2010) and bovineserum albumin (Robinson et al. 1993) in food processing, or the filtration of oilywastewater (Huotari et al. 1999), as well as for agricultural purposes, such as theseparation of protein or microbial cell suspensions (Bazinet et al. 2011; Chuang et al.2008). In addition, a phenomenon called electrolysis occurs when an aqueous solutionis subjected to an external field (Shaposhnik and Kesore 1997). The physicochemicalproperties of water are affected by its chemical composition and a number of othercomplex physical parameters, characterizing water from an energetic point of view(Kloss 1988). One of the most important parameters is the oxido-reduction potential ofwater. The reduction reaction on the cathode leads to the formation of hydroxide ions(OH−) and hydrogen gas (H2).

Cathode : 2H2Oþ 2e−→H2 þ 2OH− ð1ÞAn oxidation reaction takes place at the anode, which leads to the production of

hydronium ions (H3O+) and oxygen (O2).

Anode : 6H2O→O2 þ 4H3Oþ þ 4e− ð2Þ

This water molecular splitting occurs at standard conditions, is not thermodynam-ically attractive, and is based on the Nernst equation, which calculates 1.23 V as thestandard potential of water. Treated complex protein solutions or cell suspensionsnaturally show great differences in their salt content, depending on prior processing.However, the salt content significantly influences the effect of water molecular splitting(electrolysis) by applying electrical voltage. By subjecting a salt solution to directcurrent, electro migration of ions in the solution takes place. Positively charged ions,such as calcium and sodium (Ca2+ and Na+), move to the cathode to take up electronsand, consequently, become calcium or sodium hydroxide, while negatively chargedions, such as chloride, move to the anode, giving up electrons to become hypochloriteions, hypochlorous acid, and hydrochloric acid (Hsu 2005). As a result, molecularwater splitting is processing faster due to the ions, which again carry electrical voltage.In the case of solutions containing proteins, peptides, or amino acids, this effect mayalso influence the chemical modification of molecules to be fractionated by an electricalfield (Bazinet et al. 1997a, b; Cayot et al. 1999, 2002). In detail, hypochlorite-induced

48 A. Holder et al.

oxidation of peptides depends on the amino acid sequence of these peptides and theirreactive side chains. Cysteine (Cys) and methionine (Met) are the most reactive aminoacids interacting with hypochlorite due to their sulfur residues, followed by lysine(Lys), histidine (His), tryptophan (Trp), tyrosine (Tyr), and the R amino groups ofamino acids and peptides (Hawkins and Davies 1998; Hazell and Stocker 1993;Winterbourn 1985). In addition, some studies on hypochlorite-induced protein modifi-cation reported on protein fragmentation, side chain reactions, and aggregation thattheoretically can occur (Hawkins and Davies 1998; Hazell and Stocker 1993). Anoverview of possible responses for the α-amino groups, lysine side chains, and cysteineor methionine sulfur side chains, during cross-flow electro membrane filtration(CFEMF) experiments, is presented in Fig. 1.

However, only little is known about the impact of the electrical field during peptidefractionation via CFEMF, which is associated with the formation of hypochlorous acidthat allows hypochlorite-induced peptide modification. These chemical peptide reac-tions can, for example, lead to changes in the charge (pI) or to a fragmentation of theamino acid sequence of a peptide, which originally induces bio- or techno-functionality.These functional effects will then be lost after the chemical modification of the aminoacid sequence of these peptides. Therefore, we investigated the effects of differentelectrical field strengths on water molecular splitting in the presence of salt, lactose, andpeptides by measuring the gaseous oxygen (GO), which represents the whole processof water molecular splitting, including the formation of oxygen, hydrogen, andhypochlorous acid. In addition, the chemical modification of the highly oxidativepeptide β-CN f(108–113) and lactose, due to the interaction with hypochlorite, wasstudied in a model experiment and the impact of peptide modification during CFEMF,depending on the voltage applied, was determined.

R

NH

H C 2CO

+HOClR

NHCl

H C CO

Chloramine

-HCl-CO

OH

RNHH C 2

+H O-NH O

RH C

2

R

NH

H C

2

2

RR

NHH CR

C RH

Cross linkage

RNHCH

R

C RH

a)

NH

C CO

+HOClHS

2

b)CH2

Cysteine

NH

C COH

S

2

CH2

Sulfenylchloride

ClH

OH

OH OH

NH

C COH O

S

2

CH2

Sulfonamide

OH

O

O

Cross linkage

NH

C COH

S

2

CH2 OHSCH2

NHCH

2

CO

OH

Fig. 1 Reactions of HOCl with a α-amino groups and lysine side chains and b cysteine or methionine sulfurside chains modified according to Hawkins et al. (2003)

Electrochemical modification of peptides 49

2 Material and methods

2.1 Chemicals and raw material

All chemicals were of analytical grade. Hypochlorite and D,L-dithiothreitol werepurchased from Sigma-Aldrich (Hamburg, Germany), and NaCl, Tri-Ca phosphate,lactose monohydrate, guanidine hydrochloride, sodium citrate, and phosphate bufferwere purchased from Carl Roth GmbH (Karlsruhe, Germany). Acetonitrile (ACN,high-performance liquid chromatography (HPLC) grade, 99.9%) was obtained fromVWR International (Langendorf, Germany). Trifluoroacetic acid (TFA) and formic acid(FA) were purchased fromMerck (Darmstadt, Germany). Ultrapure water and ACN forliquid chromatography electrospray ionisation mass spectrometry (LC-ESI-MS) werepurchased from Avantor Performance Materials (Phillipsburg, NJ, USA). A micellarcasein (MCN) hydrolysate of 2.7%±0.1% dry matter and 2.0%±0.1% total protein wasused for the experiments (Holder et al. 2013a).

2.2 Electrolytic cell construction

The electrochemical treatment of water and aqueous solutions and the CFEMF ofpeptides performed thereupon were carried out using the modified filtration systemSEPA CF II from Sterlitech Corporation (WA, USA), with a membrane with amolecular weight cutoff of 5 kg.mol−1 provided by Pall Life Science (NY, USA), asdescribed previously by Holder et al. (2013b). The system was equipped to maintainconstant temperature (10 °C) by heating or cooling the feed solution in the reservoirand underwent relatively little variation (P=0.05), while transmembrane pressure wasset to 0.24 MPa. Solutions were treated for the fixed duration of 20 min with a volumeof 2.5 L. The volume flow ranged from 220 to 260 L.h−1, while the voltage used for theexperiments was adjusted in the range of 0–10 V and was generated by a DC powersupply (Model VPL-1602 PRO; Conrad Electronics, Hirschau, Germany).

2.3 Electrolysis: water molecular splitting and gas formation

The analysis of electrolysis was performed by calculating the oxido-reduction potentialvia the Nernst equation (Eq. (3)) and by measuring the gaseous oxygen (GO) evolution.

E ¼ E� þ RT

ze Flncox:cred:

ð3Þ

where E° refers to the standard cell potential, R to the universal gas constant inJoules per Kelvin per mole, T to the absolute temperature, ze to the number of moles ofelectrons transferred in the cell reaction, F to the Faraday constant in coulombs permole, and c to the chemical activity of the relevant species, where cred. is the reductantand cox. is the oxidant.

Experiments were performed without applying an electrical voltage and by applyingten different voltages: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 V (corresponding to an electricalfield strength of 333–3,333 V.m−1) at a temperature of 25 °C in order to determine theeffect of water molecular splitting on different solutions. The solutions tested were pure

50 A. Holder et al.

water, a model salt solution (including the salt and lactose content of the feed solutionduring CFEMF of peptides and is called the outer phase), and a MCN hydrolysategenerated by tryptic hydrolysis (feed solution). The composition of the outer phase wasdetermined by the Dr. Oskar-Farny-Institut (Wangen im Allgäu, Germany) andcontained 1% sodium chloride, 0.19% tricalcium phosphate, and 0.35% lactosemonohydrate.

2.4 Chemical modification

2.4.1 Model experiments of hypochlorite-induced peptide modification

The reaction of hypochlorite (HOCl) with the model peptide β-CN f(108–113), lactose,and a mixture of the peptide β-CN f(108–113) and lactose was carried out at pH 7 andon ice. The model peptide β-CN f(108–113) has been chosen because of the highreactivity of the amino acid methionine to hypochlorite, while lactose was analyzed dueto its presence in the feed solution and because of its high reaction potential withhypochlorite. Reactions were started by the addition of 1 vol HOCl reagent (commer-cial 5%) in phosphate buffer (50 mmol.L−1) to 4 vol β-CN f(108–113) with a finalconcentration of 1 mg.mL−1 of the model peptide or 1 mg.mL−1 lactose in order todetermine the impact of oxidation on the peptide and lactose. A mixture of both thepeptide and lactose was used for analyzing in a ratio of 1:6, as analyzed for the MCNhydrolysate, while HOCl was used in the range of 0.03, 3, and 30 mol.L−1. The reactionsolutions were mixed and left on ice for 15 min after the addition of HOCl.

2.4.2 Electrochemical procedure of peptide modification via CFEMF

Cross-flow electro membrane filtration of MCN hydrolysate was carried out using theelectrolytic cell, as described in Section 2.2. The fractionation of the MCN hydrolysatewas investigated with the anode located on the permeate side, using a feed solution pHof 7. Electrical voltage was set in the range of 2–10 V (667–3,333 V.m−1) in order toanalyze the impact of the chemical modification of peptides during CFEMF, as well asthe fractionation coefficient of target peptides. The wide pI range of the peptidescontained in the complex mixture of the MCN hydrolysate in combination with thefractionation parameters used (pH, polarity of electrodes, and voltage) allowed theselective fractionation of bio-functional peptides with antihypertensive properties,namely the peptides β-CN f(108–113), β-CN f(170–176), β-CN f(177–183), β-CNf(203–209), αS1-CN f(194–199), and αS2-CN f(189–197).

2.5 Evaluation of peptide modification

2.5.1 Determination of peptide modification via LC-ESI-MS

The analysis via LC-ESI-MS was performed based on Holder et al. (2014). Therefore,oxidized solutions of the peptide β-CN f(108–113) were analyzed using a nano-UPLCsystem (Acquity Waters, Milford, CT, USA) affiliated to an LTQ Orbitrap XL hybridmass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Mascot 2.3 (MatrixScience, UK) was used as the search engine for peptide identification.


2.5.2 Determination of peptides via reverse phase high performance liquidchromatography

The peptides in the feed solution and the permeate fractions of CFEMFwere determinedby reverse phase HPLC (RP-HPLC) (Holder et al. 2014). The fractionation coefficient(Si) of an antihypertensive peptide was calculated according to the following equation:

Si ¼ ci;permeateci;hydrolysate

ð4Þ

where ci,permeate refers to the concentration of an antihypertensive peptide in the perme-ate and ci,hydrolysate to the total concentration of this peptide in the MCN hydrolysate.

2.6 Evaluation of lactose modification via HPLC

The analysis of lactose was performed by HPLC using an Agilent 1100 System(Agilent Technologies, CA, USA). A 20-μL aliquot of samples was analyzed usingan ELS detector (Alltech Associates, Inc., USA) and an amino-modified polymercolumn (Prevail Carbohydrate ES, Alltech Associates, Inc., USA). Elution was carriedout using a linear gradient of 70% ACN and 30% distilled water, as published bySchuster-Wolff-Bühring et al. (2011).

2.7 Statistical analysis

All experiments were carried out in triplicate; significant differences were analyzedusing by the statistical analysis system, SAS version 9.2. Significant differences wereperformed at a probability level of P=0.05.

3 Results and discussion

3.1 Electrolysis

The oxido-reduction potentials of the solutions used were calculated via the Nernstequation, while the oxido-reduction potential for water was determined to be 1.23 V.

2H2Oþ 2e−→H2 þ 2OH− cathode; −0:83 Vð Þ6H2O→O2 þ 4H3O

þ þ 4e− anode; þ 0:40 Vð ÞX

þ1:23 V

The sodium hydroxide, firstly, has to be dissociated in water.

NaCl→Naþ þ Cl− Dissociationð ÞCathodic reaction :

4H2O→4H3Oþ þ 2OH− Autoprotolyseð Þ

2H3Oþ þ 2e−→H2 þ 2H2O

2H2Oþ 2e−→H2 þ 2OH− ECathode ¼ þ0:40 V

52 A. Holder et al.

Anodic reaction :2Cl−→Cl2 þ 2e−

2NaCl→2Naþ þ 2Cl− þ 2e− EAnodeþ−1:36 V

Furthermore, hydroxide ions react with chlorine gas to form hypochlorous acid andsodium hypochlorite.

Cl2 þ 2OH−→Cl− þ OCl− þ H2O

The electrolysis experiments were performed with a special focus on the evolutionof GO, but analogously hydrogen gas, hypochlorous acid, and all other components, asspecified by the calculation of the oxido-reduction potential, were formed during theprocess. However, the release of hydrogen gas and hypochlorous acid was not addi-tionally analyzed in these experiments. Results of electrolysis experiments at differentvoltages applied indicated that GO evolution on the anode side was significantlyaffected by the electrolysis voltage on the electrolysis cell and the salt content. Asdemonstrated in Fig. 2a, no GO was measured during the electrolysis of all threesolutions (water, outer phase, and MCN hydrolysate) within a low voltage in the rangeof 1–3 Vafter 10 min of treatment. During the electrolysis of water, only a low increasein GO in the range of 0.0043–0.007 mol was detected to be above 3 V up to 10 V. Bysubjecting the outer phase to direct current, each ion species moves according to itsown ionic mobility, depending on the electrical charge and the diffusions coefficienttoward the corresponding oppositely charged electrode. Positively charged ions, suchas calcium and sodium (Ca2+ and Na+), move to the cathode, while negatively chargedions, such as chloride, move to the anode, and thus, hypochlorite ions, hypochlorousacid, hydrochloric acid, and calcium or sodium hydroxide are generated (Fig. 3). As aresult, molecular water splitting is processing faster due to the ions, which again carryelectrical voltage.

As shown in Fig. 2a, the evolution of GO during the treatment of the MCNhydrolysate solution at various electrolysis voltages increased less than during exper-iments using the outer phase, which is due to the presence of peptides that buffer theproduction of oxygen (P=0.05). This effect was mainly observed at lower voltages inthe range of 5–7 V (corresponding to an electrical field strength of 1,667–2,333 V.m−1)compared to the outer phase. However, none of the working groups beforedifferentiated the effect of the outer phase and the feed solution, but Bolduc et al.(2006) and Bazinet et al. (2011) also reported about the effect of the oxido-reductionpotential of skimmed milk between voltages of 2–10 and 1.5–3.5 V. Bolduc et al.(2006), for example, described the optimal fractionation to be at a voltage of 4 V, whileincreasing voltage results in the formation of foam, and thus, water hydrolysis in milktakes place. The experiments performed by Bazinet et al. (2011), who worked in anarrower brand, showed that between 1.5 and 2.5 V, the effect is only low, while over alevel of 3 V, there are great differences. Compared to that, an electrical potentialbetween 8 and 16 V is sufficient enough for the production of electrolyzed water inthe food industry, which was reported by Huang et al. (2008). In addition, Fig. 2bshows the linear interrelationship of GO generation with the electrolysis amperage inboth the outer phase and MCN hydrolysate solution, which shows more or less thesame result. In the case of solutions containing salts and peptides, the development of


electrolysis and thus the formation of hypochlorite ions, hypochlorous acid, andhydrochloric acid may have a major influence on the chemical modification and the

a)

b)

Fig. 2 Oxygen (O2) evolution during electrolysis of a water (black circle), outer phase (white circle), andMCN hydrolysate solution (black down-pointing triangle) (anode on the permeate side) as a function of theelectrolysis voltage of the electrolysis cell and of b outer phase (white circle) and MCN hydrolysate solution(black down-pointing triangle) as a function of the electrolysis amperage of the electrolysis cell

Anode (+)

Cathode (-)

Permeate side

Retentate side

H O3

+

H2

Anodic reaction / Oxidation:4 OH- O2 + 2 H2O + 4 e-

6 H2 O2 + 4 H3O+ + 4 e-

Cathodic reaction / Reduction:2 H3O+ + 2 e- H2 + 2 H2O2 H2O + 2 e- H2 + 2 OH-

Cl-

Cl-

Cl-

Cl-

Cl-

Na+

Ca2+

Ca2+

H O3

+

O2

O2

H2OH

-OH-

O

Fig. 3 Oxido-reduction on the electrodes during CFEMF

54 A. Holder et al.

associated loss of bio- or techno-functional properties of peptides to be fractionated viaCFEMF (Hsu 2005; Huang et al. 2008).

3.2 Model experiments of hypochlorite-induced peptide modification

3.2.1 Determination of peptide modification via LC-ESI-MS

Experiments with hypochlorite were conducted in order to evaluate the impact ofdifferent hypochlorite concentrations on the model peptide β-CN f(108–113). Thispeptide was chosen because of the high oxidative reactivity of the amino acid methi-onine, in the amino acid sequence, against hypochlorite. Therefore, the original peptide,as demonstrated in Fig. 4a, was treated with three different hypochlorite concentrations(0.03, 3, and 30 mol.L−1), both in its pure form and in the presence of lactose. Lactoseoccurs naturally in the hydrolysate (as well as in permeate and retentate fractions) anddemonstrates high reaction potential against hypochlorite, which potentially makeslactose a peptide-protecting group. As shown in the spectrum of Fig. 4a, the β-CNf(108–113) in its pure form, as well as in the presence of lactose, remained stable at aconcentration of 0.03 mol.L−1 of hypochlorite and did not differ from the peptidefragment masses of the original peptide β-CN f(108–113) corresponding to 374.68 (z=2) and 748.36 (z=1). Compared to that, the mass spectra of the pure peptide treatedwith 3 and 30 mol.L−1 indicate differences from those of the untreated model peptideβ-CN f(108–113) (Fig. 4b, c). At a concentration of 3 mol.L−1 hypochlorite, the modelpeptide was no longer present in its original shape. Masses corresponding to the modelpeptide β-CN f(108–113) were not detected via LC-ESI-MS analysis; nevertheless,molecular masses in the range of 382.68 (z=2) and 763.33 (z=1) were detected(Fig. 4b).

Based on this result, which shows higher molecular masses of the modified peptidethan the untreated peptide, it is assumed that the main reaction during this treatmentwas the oxidation of the sulfur residue of the amino acid methionine, as shown inFig. 5. This side chain reaction was also reported by Hawkins and Davies (1998) andPattison and Davies (2001). In addition, the hypochlorite-induced oxidation on themodel peptide mixed with lactose was examined, showing the same effect on peptidemodification as the 3 mol.L−1 hypochlorite experiments with the pure β-CN f(108–113).

However, these effects were not confirmed by the experiments using concen-trations of 30 mol.L−1 hypochlorite. By increasing the concentration ofhypochlorite, not only peptide fragmentation, but mainly aggregation took place,resulting in the formation of molecular aggregates with high masses and a fewsmaller fragments up to individual amino acids (Fig. 4c). The reason for theseresults are strong reaction conditions that lead to a chain reaction of newly formedmolecules or radicals during the mechanism. Pattison and Davies (2001), whoreported that the modification of peptides, in general, depends on the amino acidsequence of the peptide and its reactive side chains, also observed these effects.The intermediate products (adducts or fragments) generated again interact with thehypochlorite or hypochlorous acid, which is an indication of further reactions.This diversity still encompasses the great complexity of the process mechanismthat is still unclear. In that case, the chemical modification of peptides by applying


200 300 400 500 600 700100

10

20

30

40

50

60

70

80

90

100

0

Inte

nsity

in%

800 900 1000

374.69

122.92

174.89212.85

310.82 488.28424.28

786.32931.68

748.36

582.69 724.01

m/z

a)

300 400 500 600 700 800m/z

0

10

20

30

40

50

60

70

80

90

100

374.69z=2

279.16488.29

z=1748.37

z=1400.92

Inte

nsity

in%

Original β-CN f(108-113)

b)

200 300 400 500 600 700100

10

20

30

40

50

60

70

80

90

100

0

Inte

nsity

in %

m/z800 900 1000

174.89

433.66

382.68

763.33

815.30

566.72310.83

702.66 899.26

200 300 400 500 600 700100

10

20

30

40

50

60

70

80

90

100

0

Inte

nsity

in %

m/z800 900 1000

158.92

180.90

307.00

429.24

278.88

494.75

585.01

618.97

698.04775.65 940.28

c)

56 A. Holder et al.

electrical voltage was considered as the sum of effects during the filtrationprocess.

3.2.2 Determination of lactose modification via HPLC

Experiments with lactose were conducted due to the high reactivity of lactose againsthypochlorite that is naturally present in the feed solution of these experiments. Theresults of the degradation of lactose indicated that an increase in the hypochloriteconcentration resulted in higher degradation of lactose than that shown via HPLC(Fig. 6). Therefore, a standard solution including the sugars fructose, glucose, galac-tose, lactulose, and lactose was used as a reference for HPLC analysis. Lactose wastreated with a concentration of 0.03 mol.L−1 hypochlorite during oxidationexperiments, resulting in a lactose degradation of 21%. Using a concentration up to3 mol.L−1 hypochlorite per milligram per milliliter of lactose, we determined adegradation of lactose which was on average 58%, while at a higher content ofhypochlorite (30 mol.L−1), the degradation content observed was calculated to be100%.

Comparing the impact of the hypochlorite concentration on lactose modification tothose of the peptide β-CN f(108–113), it can be observed that lactose processes agreater sensibility on hypochlorite than the model peptide β-CN f(108–113). Theresults indicate that the sugar lactose was oxidized firstly (starting at 0.03 mol.L−1

hypochlorite), and thus, lactose can act as a peptide-protecting group within certainlimits. Experiments with the mixed solutions containing the model peptide and lactosein a ratio of 1:6 showed a total degradation of lactose (100%) in both oxidationexperiments with 3 and 30 mol.L−1 hypochlorite, and thus, the same oxidativemodification of the model peptide was determined. These results demonstrate that theconcentration of lactose during the experiments with a ratio of 1:6 was not sufficient toprotect the peptide. However, LC-ESI-MS and HPLC seem to be reliable tools formonitoring the chemical modification of peptides by calculating the masses of frag-mentation and aggregation products, but they are not powerful enough to explain thestructural chemical formula of newly formed molecules (intermediate products) or theinformation about the process mechanism that occurs during the reaction process.Nuclear magnetic resonance (NMR) would be a possible analysis method to get moreinformation about the mechanisms of chemical reactions that took place, and thus,peptide modification.

Fig. 4 LC-ESI-MS spectra of the original β-CN f(108–113) and the hypochlorite-oxidized model peptidefragment β-CN f(108–113) in its pure form treated with a 0.03 mol.L−1 hypochlorite, b 3.0 mol.L−1

hypochlorite, and c 30 mol.L−1 hypochlorite

�

R CO

CHNH C

CS

CH3

R RCO

CHNH C

CS

CH3

+HOCl

O

R

Fig. 5 Sulfur residue oxidation of the amino acid methionine of the β-CN f(108–113)


3.3 Effects of voltage on peptide modification during CFEMF

The peptides β-CN f(108–113), β-CN f(170–176), β-CN f(177–183), β-CN f(203–209), αS1-CN f(194–199), and αS2-CN f(189–197) were determined in the permeatefraction after CFEMF via RP-HPLC and LC-ESI-MS analysis. The fractionationcoefficient of each antihypertensive peptide is given as a function of the processparameters as discussed by Holder et al. (2013b), and as a function of the electrochem-ical modification, depending on the electrical voltage applied. The electrochemicalmodification of functional peptides, if available during CFEMF, results in the loss ofthe bio- or techno-functional properties of fractionated peptides and therefore is anessential criterion of this process. However, additional design optimization of theelectro membrane filtration module can be undertaken to avoid direct contact of theproducts with the electrodes in order to reduce the risk of electrolytic reactions. Asexpected, the chemical modification of antihypertensive peptides rose with an increasein the voltage applied during CFEMF and was shown by a voltage of ≥10 V. Nomodification was observed with the experiments performed at 2–7 V. The resultsobtained for RP-HPLC and LC-ESI-MS analysis in the range of 2–7 V were compa-rable to each other in the case of electrochemical modification and provided the resultsobtained by the hypochlorite assay of the model peptide β-CN f(108–113) treated with0.03 mol.L−1 hypochlorite. At a higher voltage (10 V), side chain reactions, such assulfur residues of methionine, were detected, but no degradation or aggregation wasobserved (Fig. 1). The fractionation coefficients (Si) of the antihypertensive peptidesafter CFEMF depending on the different voltages applied are given in Table 1.

In accordance with the results from Daufin et al. (1995) which selectivelyseparated peptides and amino acids (1–9,600 V.m−1), our results show that thefractionation depends not only on the electrical field strength, but also on thephysicochemical properties of a peptide such as the charge and charge density.

Rel

ativ

e a b

sorb

ance

at

220

nm

Time in min6 1412108 16

Lac

tose

Lac

tulo

se

Glu

+ G

alFru

cto

se

a)

b)

c)

Fig. 6 HPLC chromatographic profile of the a standard sugar solution with a concentration of 0.1 g.L−1

fructose, glucose, galactose, lactulose, and lactose; b 1 mg.mL−1 lactose treated with 0.03 mol.L−1

hypochlorite; and c 1 mg.mL−1 lactose treated with 3 mol.L−1 hypochlorite

58 A. Holder et al.

Thus, low fractionation coefficients of the six antihypertensive peptides wereobtained during CFEMF for the voltages of 2–4 V applied, mainly for thepeptides β-CN f(170–176) and β-CN f(203–209) with values of about 10%,whereas the fractionation coefficients of the peptides β-CN f(177–183), β-CNf(108–113), and αS2-CN f(189–197) correspond to about 18%. Apart from thelow increase of fractionation coefficients at lower voltages, higher fractionationcoefficients of the antihypertensive peptides were observed at voltages up to7 V (corresponding to an electrical field strength of 2,333 V.m−1). This may beattributed to the effects of electrophoresis and electroosmosis (Holder et al.2013b), resulting in fractionation coefficients of about 22–26% for thepeptides β-CN f(177–183), β-CN f(108–113), and αS2-CN f(189–197), respec-tively. For the peptides β-CN f(170–176), αS1-CN f(194–199), and β-CNf(203–209), the fractionation coefficients were calculated to be in the rangeof 12–16%. In this case, no significant differences between the fractionationcoefficients of any antihypertensive peptides were obtained between voltages of5–7 V (P=0.05). Similar effects were also observed by Bargeman et al. (2002)and Poulin et al. (2006) that reported about peptide fractionation out of an α-casein hydrolysate and a β-lactoglobulin hydrolysate with migration rates be-tween 10% and 26% and by Park (2006) for the fractionation of hemoglobin.By increasing the electrical voltage up to 10 V, a decrease in the fractionationcoefficient of antihypertensive peptides (SACE) was observed (data not shown),which was also reported by Brisson et al. (2007) for the separation oflactoferrin at electrical field strengths higher than 1,667 V.m−1. Thus, it seemsthat the effect of increasing voltage during the superimposition of an electricalfield upon cross-flow membrane filtration of a complex peptide mixture resultsnot only in a higher fractionation due to the higher electrical field applied butalso in a higher chemical modification of the peptides. This leads to a lowerconcentration of the original peptides, which is in accordance to Cayot et al.(1999, 2002) and Hawkins et al. (2003).

Based on this information, the optimum fractionation condition needs to be deter-mined, enabling both high peptide fractionations connected with low chemical

Table 1 Fractionation coefficients of the six antihypertensive peptides in the CFEMF permeate after electricalvoltages applied in the range of 2–7 V

Peptide Si of target peptides in % at electrical voltages

2 V 3 V 4 V 5 V 6 V 7 V

β-CN f(108–113) 16.5±1.0 18.0±0.7 15.3±1.1 25.5±1.1 26.8±1.3 26.2±0.6

β-CN f(177–183) 17.9±0.6 17.7±0.8 19.4±1.2 20.8±0.3 21.9±0.3 22.1±0.3

β-CN f(170–176) 11.3±0.6 9.6±1.2 8.9±0.7 12.1±0.9 15.2±0.5 15.6±0.4

αS2-CN f(189–197) 17.6±0.4 17.8±0.1 17.7±0.3 19.9±1.2 21.9±0.8 22.9±0.4

αS1-CN f(194–199) 13.0±0.5 12.0±0.3 12.8±0.7 17.3±0.5 15.8±0.6 16.4±1.1

β-CN f(203–209) 9.3±0.7 10.0±0.3 10.1±0.4 11.6±0.2 11.5±0.5 11.8±0.2

ΣSACE 11.6±1.1 12.5±0.4 12.6±0.9 14.5±0.5 15.0±0.7 15.1±0.6


modification of peptides, in order to achieve the application of functional peptides withbio- or techno-functional properties for the food industry. However, from an energeticpoint of view, a voltage of 5 V (1,667 V.m−1) is sufficient for the fractionation ofpeptides due to only insignificantly lower fractionation coefficients of the peptides thanby a voltage of 7 V (2,333 V.m−1) applied. In addition, LC-ESI-MS showed nochemical modification of peptides at a voltage of 5 V, while GO evolution thataccompanies the formation of hypochlorous acid was low for the MCN hydrolysateat a voltage of 5 V.

4 Conclusion

In conclusion, the effects of electrolysis, electro modification, and electropho-resis of dairy-based functional peptides were determined. We have demonstratedthe impact of the electrical field during peptide fractionation on the electro-chemical modification of functional peptides. The electrolysis experiments withthe MCN hydrolysate solution showed a minimum of water molecular splittingand gas formation in the range of 4–5 V, while the formation of oxygen andthe associated formation of hydroxide gas and hypochlorous acid for voltagesof ≥6 V significantly increased in these experiments.

The electrochemical modification of the model peptide β-CN f(108–113) wasdetermined in a hypochlorite oxidation assay. An increase in reaction productsincluding aggregates and decomposition products down to the level of singleamino acids was found by LC-ESI-MS of solutions treated with a high hypo-chlorite concentration of 30 mol.L−1. The more complex structural modificationof the MCN hydrolysate during CFEMF to adducts or degradation products wasdetermined in sum, in relation to the fractionation coefficients of the sixantihypertensive peptides. Although high voltage has advantages in thefractionation effect of these peptides, voltage should be kept low for a carefulhandling of products generated for further food processing. An electrical fieldof 1,666 V.m−1 (5 V) permits the fractionation of high concentrations ofantihypertensive peptides without limiting the process due to electrochemicalmodification, taking into account only low gaseous evolution resulting fromelectrolysis. As a general strategy for the industrial upscale of the superimpo-sition of an electrical field upon cross-flow membrane filtration, our findingssuggest examination of the effect of an electrical field applied with electricalfield strengths of <2,333 V.m−1, depending on the feed solution used. In thisconstellation, the application of cross-flow electro membrane filtration has theadvantages of low energy costs per gram of fractionated peptide compared toconventional filtration and the process can easily be scaled up at industrialscale. In addition, there is no modification of the composition of the feedsolution during cross-flow electro membrane filtration, as it is currently thecase of chromatographic processes, enabling an uncomplicated application offractionated peptides in food products.

Acknowledgments This research project was supported by the German Ministry of Economics andTechnology (via AiF) and the FEI (Forschungskreis der Ernährungsindustrie e.V., Bonn) Project AiF 16541N.

60 A. Holder et al.

Conflict of interest Aline Holder, Lutz Großmann, Jochen Weiss, and Jörg Hinrichs declare that they haveno conflict of interest.

References

Bargeman G, Koops GH, Houwing J, Breebaart I, Van Der Horst HC,Wessling M (2002) The development ofelectro-membrane filtration for the isolation of bioactive peptides: the effect of membrane selection andoperating parameters on the transport rate. Desalination 149(1–3):369–374

Bazinet L, Lamarche F, Labrecque R, Ippersiel D (1997a) Effect of KCl and soy protein concentrations on theperformance of bipolar membrane electroacidification. J Agric Food Chem 45(7):2419–2425

Bazinet L, Lamarche F, Labrecque R, Ippersiel D (1997b) Effect of number of bipolar membranes andtemperature on the performance of bipolar membrane electroacidification. J Agric Food Chem 45(10):3788–3794

Bazinet L, Schreyer A, Lessard J (2011) Optimization of milk electroreduction: impact of low anode/cathodevoltage difference application on its redox potential modulation during treatment and storage. Dairy SciTechnol 91(5):525–540

Bolduc MP, Bazinet L, Lessard J, Chapuzet JM, Vuillemard JC (2006) Electrochemical modification of theredox potential of pasteurized milk and its evolution during storage. J Agric Food Chem 54(13):4651–4657

Brisson G, Britten M, Pouliot Y (2007) Electrically-enhanced crossflow microfiltration for separation oflactoferrin from whey protein mixtures. J Membr Sci 297(1–2):206–216

Cayot P, Roullier L, Tainturier G (1999) Electrochemical modifications of proteins. 1. Glycitolation. J AgricFood Chem 47(5):1915–1923

Cayot P, Rosier H, Roullier L, Haertlé T, Tainturier G (2002) Electrochemical modifications of proteins:disulfide bonds reduction. Food Chem 77(3):309–315

Chuang C-J, Wu C-Y, Wu C-C (2008) Combination of crossflow and electric field for microfiltration ofprotein/microbial cell suspensions. Desalination 233(1–3):295–302

Daufin G, Kerhervé FL, Aimar P, Mollé D, Léonil J, Nau F (1995) Electrofiltration of solutions of amino acidsor peptides. Lait 75(2):105–115

Hawkins CL, Davies MJ (1998) Hypochlorite-induced damage to proteins: formation of nitrogen-centredradicals from lysine residues and their role in protein fragmentation. Biochem J 332(3):617–625

Hawkins CL, Pattison DI, Davies MJ (2003) Hypochlorite-induced oxidation of amino acids, peptides andproteins. Amino Acids 25(3–4):259–274

Hazell LJ, Stocker R (1993) Oxidation of low-density lipoprotein with hypochlorite causes transformation ofthe lipoprotein into a high-uptake form for macrophages. Biochem J 290(1):165–172

Hofmann R, Posten C (2003) Improvement of dead-end filtration of biopolymers with pressureelectrofiltration. Chem Eng Sci 58(17):3847–3858

Holder A, Birke A, Eisele T, Klaiber I, Fischer L, Hinrichs J (2013a) Selective isolation of angiotensin-I-converting enzyme-inhibitory peptides from micellar casein and β-casein hydrolysates via ultrafiltration.Int Dairy J 31(1):34–40

Holder A, Scholz S, Kulozik U, Hinrichs J (2013b) Cross-flow electro membrane filtration: theory andapplication in the dairy industry. Chem Ing Tech 85(8):1193–1200

Holder A, Thienel K, Klaiber I, Pfannstiel J, Weiss J, Hinrichs J (2014) Quantification of bio- and techno-functional peptides in tryptic bovine micellar casein and β-casein hydrolysates. Food Chem 158:118–124

Hsu SY (2005) Effects of flow rate, temperature and salt concentration on chemical and physical properties ofelectrolyzed oxidizing water. J Food Eng 66(2):171–176

Huang YR, Hung YC, Hsu SY, Huang YW, Hwang DF (2008) Application of electrolyzed water in the foodindustry. Food Control 19(4):329–345

Huotari HM, Huisman IH, Trägårdh G (1999) Electrically enhanced crossflow membrane filtration of oilywaste water using the membrane as a cathode. J Membr Sci 156(1):49–60

Käppler T, Posten C (2007) Fractionation of proteins with two-sided electro-ultrafiltration. J Biotechnol128(4):895–907

Kloss AI (1988) Electron-radical dissociation and mechanism of water activation. Trans Acad Sci USSR 303:1403–1406

Lentsch S, Aimar P, Orozco JL (1993) Enhanced separation of albumin-poly (ethylene glycol) by combinationof ultrafiltration and electrophoresis. J Membr Sci 80:221–232


Ndiaye N, Pouliot Y, Saucier L, Beaulieu L, Bazinet L (2010) Electroseparation of bovine lactoferrin frommodel and whey solutions. Sep Purif Technol 74(1):93–99

Park YG (2006) Effect of an electric field during purification of protein using microfiltration. Desalination191(1–3):404–410

Pattison DI, Davies MJ (2001) Absolute rate constants for the reaction of hypochlorous acid with protein sidechains and peptide bonds. Chem Res Toxicol 14(10):1453–1464

Poulin JF, Amiot J, Bazinet L (2006) Simultaneous separation of acid and basic bioactive peptides byelectrodialysis with ultrafiltration membrane. J Biotechnol 123(3):314–328

Robinson CW, Siegel MH, Condemine A, Fee C, Fahidy TZ, Glick BR (1993) Pulsed-electric-field crossflowultrafiltration of bovine serum albumin. J Membr Sci 80:209–220

Schuster-Wolff-Bühring R, Michel R, Hinrichs J (2011) A new liquid chromatography method for thesimultaneous and sensitive quantification of lactose and lactulose in milk. Dairy Sci Technol 91(1):27–37

Shaposhnik VA, Kesore K (1997) An early history of electrodialysis with permselective membranes. J MembrSci 136(1–2):35–39

Winterbourn CC (1985) Comparative reactivities of various biological compounds with myeloperoxidase-hydrogen peroxide-chloride, and similarity of the oxidant to hypochlorite. Biochim Biophys Acta GenSubj 840(2):204–210

62 A. Holder et al.

electrochemical modification of dairy-based functional

Documents