foaming properties of tryptic gliadin

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Foaming properties of tryptic gliadin hydrolysate peptide fractions

Bert G. Thewissen, Inge Celus, Kristof Brijs , Jan A. Delcour

Laboratory of Food Chemistry and Biochemistry, Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven, Kasteelpark

Arenberg 20, B-3001 Leuven, Belgium

a r t i c l e i n f o

Article history:

Received 22 September 2010

Received in revised form 10 January 2011Accepted 1 March 2011

Available online 5 March 2011

Keywords:

Foaming properties

Gliadin

Hydrolysates

Peptide fractions

a b s t r a c t

A tryptic gliadin hydrolysate was separated into central domain (CD) or terminal domain (TD) related

peptide fractions. Whereas the initial foam volume (FV) of CD peptide fractions remained constant as a

function of pH, FV of TD peptide fractions increased from acidic to alkaline pH. Foam stability (FS) of

CD peptide fractions was maximal near neutral pH. For TD peptide fractions, one fraction showed max-

imal FS at strongly alkaline pH, while the other showed no clear maximal FS. CD related peptide foams

contained higher levels of hydrophobic peptides than the respective solutions, while small differences

were observed for TD peptide fractions. Peptide compositions of foams did not vary with pH, indicating

that the foaming properties of gliadin peptides are mainly dictated by charges. As the pH dependent

foaming properties of TD related peptides resemble best those of gliadin, it was concluded that the pH

dependent foaming properties of gliadins are mainly determined by their TDs.

2011 Elsevier Ltd. All rights reserved.

1. Introduction

Commercial wheat gluten is the protein rich co-product of

industrial wheat starch isolation. It consists of comparable

amounts of gliadin and glutenin. Its visco-elastic properties after

hydration are one of its predominant features. The gliadin fraction

of wheat gluten is poorly soluble near neutral pH (Thewissen, Ce-

lus, Brijs, & Delcour, 2011), which limits its applicability in a lot of

food systems. On the other hand, gliadins show excellent foaming

properties near neutral and at alkaline pH. Foaming properties are

rather poor at acidic pH (Mita, Ishida, & Matsumoto, 1978; Thewis-

sen et al., 2011). In the previous work (Thewissen et al., 2011), it

was reported that gliadin foams at acidic and alkaline pH are selec-

tively enriched inc-gliadins. In contrast, the levels ofa- and c-gli-

adins in gliadin foams were similar at pH 8.0.Uthayakumaran et al.

(2001)found c-gliadins to have higher foaming stability than the

other gliadin types. Our group established that positively charged

amino acids (AA) lead to electrostatic repulsion between gliadins,

hindering foam stabilisation at acidic pH (Thewissen et al., 2011).

Addition of chloride ions, shielding these positive charges at acidic

pH, improved foaming properties.

In order to improve the water solubility and foaming properties

over a wider pH range, gliadin can be enzymatically hydrolysed. Tothe best of our knowledge, no research efforts have been made on

the foaming properties of gliadin hydrolysates. In contrast, litera-

ture reports on the foaming properties of gluten hydrolysate mix-

tures. Hydrolysis of gluten leads to peptides that either have higher

or lower foaming properties than the parent material.Linars, Lar-

r, Lemeste, and Popineau (2000)reported both increased foaming

capacity and foam stability (FS) with an increasing degree of

hydrolysis (DH). In contrast, Drago and Gonzalez (2001) observed

only decreased foaming capacity and FS with increasing DH.Kong,

Zhou, and Qian (2007)found increased foaming capacity and FS at

low DH, while foaming capacity and FS decreased with increasing

DH.

Also, pH affects the foaming properties of gluten hydrolysates.

Drago and Gonzalez (2001) showed that the foaming capacity

and FS increases from pH 4.0 to 9.0. In contrast, Popineau, Huchet,

Larr, and Brot (2002) reported better foaming properties at pH

4.0 than at pH 6.5. Wang, Zhao, Bao, Hong, and Rosella (2008)

found no differences in foaming capacity of gluten hydrolysates be-

tween pH 5.0, 7.0 and 8.0. Hydrophobic gluten peptides, containing

most of the ionisable AA, show higher foaming capacity and FS

than hydrophilic peptides (Brot, Popineau, Compoint, Blassel, &

Chaufer, 2001; Popineau et al., 2002).

This research aims to investigate the foaming properties of

gliadin hydrolysates and to relate these properties to the foaming

properties of gliadin (Thewissen et al., 2011). More in particular,

the objectives are to improve the solubility of gliadin by enzymatic

hydrolysis and to examine and understand the impact of pH on the

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.doi:10.1016/j.foodchem.2011.03.007

Abbreviations:AA, amino acid(s); CD, central domain(s); db, on dry basis; DH,

degree of hydrolysis (%); FS, foam stability; FV, initial foam volume; MW, molecular

weight; PES, polyethersulfone; pI, isoelectric point; RP-HPLC, reversed-phase HPLC;

SE-HPLC, size-exclusion HPLC; Tr, room temperature (C); (C-N-) TD(s), (C-N-)

terminal domain(s); TFA, trifluoroacetic acid. Corresponding author. Tel.: +32 0 16321634; fax: +32 0 16321997.

E-mail addresses: [email protected] (I. Celus), [email protected]

leuven.be(K. Brijs), [email protected](J.A. Delcour).

Food Chemistry 128 (2011) 606612

Contents lists available at ScienceDirect

Food Chemistry

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http://dx.doi.org/10.1016/j.foodchem.2011.03.007mailto:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.03.007http://www.sciencedirect.com/science/journal/03088146http://www.elsevier.com/locate/foodchemhttp://www.elsevier.com/locate/foodchemhttp://www.sciencedirect.com/science/journal/03088146http://dx.doi.org/10.1016/j.foodchem.2011.03.007mailto:[email protected]:[email protected]:[email protected]:[email protected]://dx.doi.org/10.1016/j.foodchem.2011.03.007


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foaming properties of structural different gliadin peptides. The pri-

mary structure of gliadins indeed consists of a hydrophilic central

domain (CD) containing repetitive AA sequence particularly rich in

Gln and is enclosed by two more hydrophobic terminal domains

(TDs) containing low levels of Gln and Pro, and higher levels of

hydrophobic AA than the CD. In addition, almost all ionisable AA,

which are present in low levels, occur in the C-TD (Shewry &

Tatham, 1990). In the long run, this research can contribute toinsights in the role of gliadin and gliadin hydrolysates in wheat

based aerated food systems, such as in bread and cakes.

2. Experimental

2.1. Materials

Commercial wheat gluten [protein content (N 5.7): 80.22% on

dry basis (db)] was from Cargill (Bergen op Zoom, The Nether-

lands). Trypsin from porcine pancreas was from SigmaAldrich

(Bornem, Belgium). Denatured ethanol (97% v/v) was from Brenn-

tag (Mlheim/Ruhr, Germany). All chemicals, solvents and re-

agents were purchased from SigmaAldrich and were of

analytical grade unless otherwise specified.

2.2. Methods

2.2.1. Gliadin hydrolysis and fractionation of the resulting peptide

mixture

Tryptic gliadin hydrolysis was performed as described by The-

wissen, Celus, Brijs, and Delcour (2010)and adapted to larger scale

production. Gliadin was extracted in two steps from commercial

wheat gluten (100 g) with 70% (v/v) aqueous ethanol solution

(1.00 l). The ethanol in the supernatant was removed by rotary

evaporation (50 C) and the gliadin fraction freeze-dried. A volume

of 1.00 l of a 6.0% (wprotein/v) aqueous dispersion of wheat gliadin

was incubated with 5.0% (w/wprotein) trypsin (pH 8.0; 50C;

3.0 h). The pH was kept at pH 8.0 by manual addition of 2.0 MNaOH. Afterwards, the mixture (GliaTryptotal) was adjusted to pH

6.0 with 2.0 M HCl and heated at 90 C for 15 min to inactivate

the enzyme. It was centrifuged (10.0 min; 10,000g; 20 C) and both

supernatant (GliaTrypsol) and residue (GliaTrypinsol) were freeze-

dried. GliaTrypsol was further fractionated by graded ethanol

precipitation as described byThewissen et al. (2010). To this end,

GliaTrypsol (6.0% wprotein/v) was suspended in deionised water

and aliquots of ethanol were added to the protein solution under

continuous stirring to obtain a final ethanol concentration of 80%.

The mixture was then kept overnight at 4 C. Precipitated material

(GliaTrypsol0-80) was recovered by centrifugation (10,000g;

10.0 min; 4C), suspended in deionised water and freeze-dried.

The ethanol concentration in the supernatant was further in-

creased to 90% (v/v) and the precipitated material (GliaTrypsol

80-

90) was recovered as described before. Ethanol was removed from

the remaining supernatant (GliaTrypsol90+) by rotary evaporation

(50C).

2.2.2. Chemical composition of GliaTryp fractions

2.2.2.1. Protein contents. Protein contents were determined using

the Dumas combustion method, an adaptation of the AOAC official

method 990.03 (AOAC, 1995) to an automated Dumas protein anal-

ysis system (EAS, varioMax N/CN, Elt, Gouda, The Netherlands),

using 5.7 as the conversion factor for gluten proteins.

2.2.2.2. Amino acid composition. Amino acid (AA) composition was

determined following release by acid hydrolysis as described by

Rombouts et al. (2009). AA were separated by anion-exchange highperformance liquid chromatography with AminoPac PA10 guard

(50 2 mm) and analytical (250 2 mm, Dionex, Sunnyvale, CA,

USA) columns using a Dionex BioLC system equipped with a

GS50 gradient pump, an AS50 autosampler and an ED50 electro-

chemical detector. During acid hydrolysis, Gln and Asn are trans-

formed into Glu and Asp, respectively.

2.2.2.3. Ash contents. Ash contents were determined according to

AACC method 08-12 (AACC, 2000).

2.2.2.4. Monosaccharide compositions. Monosaccharide composi-

tions of gliadin and GliaTryp fractions were determined by the

method ofEnglyst and Cummings (1984).

2.2.2.5. Solubility of gliadin in water. Prior to foam formation, gliadin

peptides were dispersed in deionised water (0.3% w/v protein). The

pH of the suspensions was adjusted to pH 2.0, 6.7, 8.0, 8.0 and 12.0

with either 1.0 M HCl or 1.0 M NaOH. Gliadin peptide suspensions

were also prepared in the presence of 2.00% (w/v) NaCl. Protein

contents in supernatants (further referred to as peptide solutions),

obtained after centrifugation (10,000g; 10 min; 20 C) of the sus-

pensions, were determined by the above mentioned Dumas meth-

od, using 5.7 as the nitrogen to protein conversion factor for gluten

proteins.

2.2.3. Foaming properties

Foams were prepared based on the whipping method ofCaes-

sens, Gruppen, Visser, van Aken, and Voragen (1997) with small

modifications. A volume of 100 ml of peptide solution was placed

in a graduated glass cylinder (internal diameter: 60 mm), of which

the bottom was covered with a glass filter (thickness: 5 mm; diam-

eter: 60 mm) and had a small tap to allow removal of the aqueous

liquid phase (Thewissen et al., 2011). The solution was whipped for

70 s using a rotating propeller (2000 rotations per minute; outer

diameter: 45.0 mm; thickness: 0.4 mm) at room temperature (Tr).

The initial foam volume (FV, ml) was that measured 2.0 min after

the start of whipping. Foam volume loss was monitored during

60 min and FS (%) was defined as the percentage of FV remainingafter 60 min relative to FV. After 60 min, the liquid under the foam

was removed through the tap, while the residual foam on top of

the glass filter was removed and recovered with 70% (v/v) aqueous

ethanol solution. The peptide solutions, the remaining aqueous

solution after foam formation and the resulting foams were

freeze-dried. The coefficient of variation for the determination of

FV and FS was calculated based on a fivefold determination with

a typical sample and did not exceed 10%.

2.2.4. Surface tension measurements

Surface tensions (Nm1) of peptide solutions were determined

atTrusing a torsion balance (model OS Balance/Tensiometer, Bid-

ford on Avon, Alcester, Warwickshire, UK) equipped with a

40.0 mm circumference platinum (DuNuoy) ring. Recipients con-taining the gliadin peptide solutions were cleaned with acetone

and air-dried before use. The coefficient of variation for surface

tension values was calculated based on a fivefold determination

of a typical sample solution and did not exceed 1.0%.

2.2.5. Reversed-phase HPLC

The distribution of gliadin peptides in initial peptide solutions

and foams were determined by reversed-phase HPLC (RP-HPLC).

Peptide samples were dissolved in 70% aqueous ethanol solution

(0.5% wprotein/v), filtered through a 0.45 lm polyethersulfone

(PES) membrane (Millipore, Billerica, MA, USA) and injected

(40 ll) on a Vydac 201TP C18 column (5 lm, 250 3.0 mm, All-

tech Associates, Deerfield, IL, USA) at 50 C using a LC-2010 HPLC

system (Shimadzu, Kyoto, Japan) with automated sample injection.The elution solvent consisted of milli-Q water (solvent A) and ace-

B.G. Thewissen et al./ Food Chemistry 128 (2011) 606612 607


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tonitrile (ACN) (solvent B), both containing 0.1% (v/v) trifluoroace-

tic acid (TFA). Peptides were eluted with a linear gradient from 0%

to 100% solvent B in 160 min at a flow rate of 0.5 ml/min and de-

tected by absorbance detection at 214 nm.

2.2.6. Size-exclusion HPLC

Size-exclusion HPLC (SE-HPLC) was performed with a Biosep-

SEC-S 2000 column (300 x 7.8 mm, Phenomenex, Torrance, CA,USA) using a Shimadzu LC-2010 system with automated sample

injection (Thewissen et al., 2010). The elution solvent was ACN/

milli-Q water (1:1, v/v) including 0.05% (v/v) TFA. Samples (0.1%

wprotein/v) were dissolved in the elution solvent and filtered

through a 0.45 lm PES membrane (Millipore). The injection vol-

ume was 60 ll, the flow rate 1.0 ml/min, and the temperature

30C. Peptides were detected by absorbance detection at

214 nm. The column was calibrated with the molecular weight

(MW) markers ribonuclease A (13.7 k), aprotinin (6.5 k), (Ala)5(373) and AlaGln (217).

3. Results and discussion

3.1. Fractionation and characterisation of CD and TD related peptide

fractions

The gliadin fraction, extracted with a 70% ethanol solution from

wheat gluten, was hydrolysed with trypsin to obtain the tryptic gli-

adin peptide mixture (Thewissen et al., 2010). Based on their AA

compositions, GliaTrypsol0-80 and GliaTrypsol80-90 are related to

the CD, whereas GliaTrypsol90+ and GliaTrypinsolare related to the

gliadin TDs (Thewissen et al., 2010).

The highest MW peptides were found in GliaTryp insol, followed

by those in GliaTrypsol0-80, GliaTrypsol80-90 and GliaTrypsol90+

(SE-HPLC results not shown). RP-HPLC analysis showed that Glia-

Trypsol90+ peptides eluted first (most hydrophilic conditions), fol-

lowed by those of GliaTrypsol80-90, GliaTrypsol0-80 and

GliaTrypinsol (most hydrophobic conditions), respectively (resultsnot shown). These results are in line with those of similar work

on a smaller scale (Thewissen et al., 2010). This was expected as

the process conditions (protein concentration, enzyme to substrate

ratio, pH and temperature) at which hydrolysis was performed,

were identical.

Table 1lists the results of a partial chemical analysis of the dif-

ferent GliaTryp fractions. Protein yields are defined as the ratio of

protein weight of a peptide fraction to the protein weight of Glia-

Tryptotal, and expressed as a percentage. The soluble peptides (Gli-

aTrypsol) represent 86% of GliaTryptotal. After graded ethanol

precipitation of GliaTrypsol, almost half of the soluble peptide frac-

tion was recovered in GliaTrypsol0-80 (Table 1). The protein con-

tents of GliaTrypsol0-80 and GliaTrypsol80-90 were very high [99%

and 93% (w/w, db), respectively], while those of GliaTrypsol90+

and GliaTrypinsolwere lower (54% and 56%, respectively). The levels

of arabinose, xylose and mannose were very low. Arabinose and

xylose probably originate from arabinoxylan. Higher levels of glu-

cose were present in GliaTrypsol90+. Glucose probably originates

from dextrins, which are liberated from starch. GliaTrypsol90+

and GliaTrypinsolalso contained substantial levels of galactose, that

probably originated from galactolipids associated with gluten(Bks, Zawistowska, & Bushuk, 1983).

3.2. Solubility of GliaTryp fractions as a function of pH

Peptides of GliaTrypsol80-90 and GliaTrypsol90+ were almost

completely soluble at pH 2.0, 6.7, 8.0 and 12.0 ( Fig. 1A). The solu-

bility of GliaTrypsol0-80 exceeded 90% at pH 2.0 and 6.7, while at

pH 8.0 and 12.0, solubility was about 75%. While GliaTrypinsolwas separated from GliaTrypsol at pH 6.0 by centrifugation, more

than 60% of the proteins were soluble at pH 2.0, 6.7 and 8.0 and sol-

ubility was complete at pH 12.0.

3.3. Foaming properties of GliaTryp fractions

3.3.1. General

The foaming properties of solutions of GliaTryp fractions were

determined at pH 2.0, 6.7, 8.0 and 12.0 without correcting for the

variability in protein concentration, which itself resulted from a

varying solubility of the peptides as a function of pH.

3.3.2. CD related peptide fractions

FV of CD related peptide fractions (GliaTrypsol0-80 and Glia-

Trypsol80-90) were rather constant (60 ml) at pH 2.0, 6.7, 8.0

and 12.0, except for GliaTrypsol80-90 at pH 6.7, which, under the

experimental conditions, had a FV exceeding 70 ml (Fig. 2A and

B). Although FV of CD related peptide fractions were rather con-

stant, FS of both fractions varied as a function of pH. Maximal FS

values for GliaTrypsol0-80 and GliaTrypsol80-90 were obtained atpH 6.7 and 8.0, respectively, while FS decreased towards more

acidic and alkaline pH. In both cases, FS was lower at pH 12.0 than

at pH 2.0.

At pH below 8.0, both peptide fractions showed similar FV as

native gliadin, while, at pH 12.0, FV was slightly lower than for na-

tive gliadin (Fig. 2) (Thewissen et al., 2011). The overall lower FS of

these peptide fractions than those of native gliadins is probably

due to the lower MW of peptides leading to less interactions at

the airwater interface (Foegeding, Luck, & Davis, 2006). In con-

trast, at pH 2.0, CD related peptide fractions, and, in particular,

GliaTrypsol0-80, showed higher FS than native gliadin (Thewissen

et al., 2011). This can be explained by the fact that most ionisable

AA are present in the TD of gliadins, which means that the CD

Table 1

Partial chemical composition (% db) and protein yield of the tryptic gliadin peptide fractions.

Fraction Protein yield (%)a Protein content (%)b Ash (%) Carbohydrates (%)

Glucose Galactose Arabinose Xylose Mannose

Gliadin 100 85 1.2 5.07 1.55 0.13 0.05 0.45

GliaTrypsol 86 85 n.d. n.d. n.d. n.d. n.d. n.d.

GliaTrypsol0-80 46 99 1.3 1.43 0.51 0.12 0.07 0.17

GliaTrypsol 80-90 20 93 1.6 2.84 0.80 0.04 0.05 0.61

GliaTrypsol 90+ 20 54 15.1 19.75 7.33 0.09 0.03 0.84

GliaTrypinsol 14 56 1.7 1.15 6.22 0.12 0.00 0.16

n.d, not determined.a

% (w/w) of protein weight in the respective fraction to the total protein weight before fractionation.b N 5.7.

608 B.G. Thewissen et al. / Food Chemistry 128 (2011) 606612


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related peptide fractions contain lower levels of AA with ionisableside chains, resulting in less electrostatic repulsion.

3.3.3. TD related peptide fractions

FV of TD related peptide fractions (GliaTrypsol90+ and Glia-

Trypinsol) increased from acidic to alkaline pH (Fig. 2C and D). At

each particular pH, FV of GliaTrypsol90+ was lower than for Glia-

Trypinsol. No residual foam was left 60 min after the start of whip-

ping of GliaTrypsol90+ solution at pH 2.0, while FS was only about

30% at the other pH values. In contrast, the FS of GliaTryp insol at

acidic pH was similar to that at alkaline pH and higher than thatof GliaTrypsol90+. The lower FS of GliaTrypsol90

+ than that of Glia-

Trypinsol could be explained by their lower MWs leading to less

intermolecular interactions at the interface, which are crucial for

stable foams (Foegeding et al., 2006). The FS of GliaTrypinsol was

higher at pH 2.0 than for native gliadin (Thewissen et al., 2011)

but lower at higher pH.

The poorer foaming properties of TD related peptide fractions at

more acidic pH than those of CD related peptide fractions can be

explained by the presence of higher levels of Lys and Arg residues.

As a result, the isoelectric point (pI) of TD related peptide fractions

is probably higher, resulting in more electrostatic repulsion be-

tween TD related peptides at acidic pH at the interface. In silico

analysis of a tryptic digest of gliadin indeed showed that TD related

peptides show a higher pI (results not shown).

The pH dependent foaming properties of TD related peptides

resemble those of gliadin (Thewissen et al., 2011), while those of

CD related peptides do not. Therefore, we believe that the pH

dependent foaming properties of gliadins are mainly dictated by

their TD.

3.4. Surface tension of GliaTryp fractions

Due to cohesive (van der Waals) interactions between the mol-

ecules at airliquid interfaces, they have a surface tension. Sub-

stances with amphiphilic properties can adsorb at airliquid

interfaces thereby decreasing the surface tension, which, after

foam formation, results in foams with increased kinetic stability

(Patino, Sanchez, & Nino, 2008). In order to relate the surface-ac-tive properties of solutions of CD and TD related gliadin peptide

A

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

2 .0 6.7 8.0 12.0

Solubilise

dprotein(%,w/v)

pH

B

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

2.0 6.7 8.0 12.0

Solubilisedp

rotein(%,w/v)

pH

Fig. 1. Solubilised protein (%, w/v) of GliaTrypsol0-80 (j), GliaTrypsol80-90 ( ),

GliaTrypsol90+ ( ) and GliaTrypinsol( ) dispersions (0.3% w/v) at pH 2.0, 6.7, 8.0 and

12.0, set at pH using 1.0 M HCl or NaOH (A) without or (B) in the presence of 2.0%

NaCl.

A B

C

0

20

40

60

80

100

2.0 6.7 8.0 12.0

FV(mL),FS(%)

pH

0

20

40

60

80

100

2.0 6.7 8.0 12.0

FV(mL),FS(%)

pH

0

20

40

60

80

100

2.0 6.7 8.0 12.0

FV(mL),FS(%)

pH

0

20

40

60

80

100

2.0 6.7 8.0 12.0

FV(mL),FS(%)

pH

D

Fig. 2. Initial foam volume (FV, solid) and foam stability (FS, transparent) 1.0 h after foam formation of GliaTryp sol0-80 (A), GliaTrypsol8090 (B), GliaTrypsol90+ (C) andGliaTrypinsol (D) solutions at pH 2.0, 6.7, 8.0 and 12.0, set at pH using 1.0 M HCl or NaOH without (black) or in the presence of 2.0% NaCl (grey).



5/7

fractions at different pHs to their corresponding foaming proper-

ties, the surface tensions of gliadin peptide solutions were deter-

mined (Fig. 3).

The surface tensions of the CD related peptide fractions varied

little as a function of pH (Fig. 3). This was in line with their con-

stant FVs as function of pH. The surface tension values of TD re-

lated peptide solutions (GliaTrypsol90+ and GliaTrypinsol) were

lower than those of their CD counterparts (GliaTrypsol0-80 and Gli-aTrypsol80-90) at all pH values tested. However, TD related peptide

fractions had no better foaming properties than CD related peptide

fractions. The surface tensions of the TD related fractions varied

with pH. Higher surface tensions were measured for GliaTrypsol90+

at alkaline pH. However, their FVs increased with pH. The surface

tensions of GliaTrypinsol solutions were slightly lower at pH 8.0

than at more extreme pH values. In general, the constant surface

tensions as a function of pH of CD related peptide solutions were

in line with their constant FV. There was no link between the sur-

face tension and FV for the TD related peptides.

3.5. Peptide distribution in foams from GliaTryp peptide fractions

RP-HPLC discriminated between the peptides in foams, remain-ing 60 min after the start of whipping, and the remaining solution.

It is based on differences in hydrophobicity, although MW also

influences separation by RP-HPLC. The resulting chromatograms

were subdivided into four randomly chosen areas (5.032.0 ml,

32.049.0 ml, 49.059.0 ml, 59.0125.0 ml elution volumes), fur-

ther referred to as RP fractions A, B, C and D, respectively. The per-

centage area of each RP fraction was expressed relatively to the

total area of the RP chromatogram.

Foams of CD related fractions were clearly enriched in hydro-

phobic peptides (RP fractions C and D) compared to the respective

solutions (Fig. 4A,B,E and F). In contrast, only small differences

were observed between the distribution of peptides present in

foam and solution from TD related peptide fractions (Fig. 4C,D,G

and H). Although the foaming parameters differ according to thepH, none of the GliaTryp fractions showed differences in peptide

distributions within both foams and remaining solutions at varying

pH. A possible explanation is that charges on the peptides at a cer-

tain pH are responsible for the pH dependent foaming properties

rather than differences in peptide distribution within the foams,

as the latter did not vary. This was also the conclusion of earlier

work on the foaming properties of gliadin (Thewissen et al.,

2011). In general, at pH values that are different from their pI, pep-

tides carry a net charge which results in electrostatic repulsion be-

tween peptides at the interface, so that less peptides adsorb at theinterface and less peptide-peptide interactions occur, resulting in a

decreased FS (Foegeding et al., 2006; Patino et al., 2008). CD related

peptide fractions contain lower levels of Lys and Arg than TD re-

lated peptide fractions (Thewissen et al., 2010), which may indi-

cate that the pI of TD related peptides is higher than that for CD

related peptides. This may explain the poor foaming properties of

TD related peptide fractions at more acidic pH while CD related

peptide fractions show constant FV as a function of pH and optimal

FS at lower pH (near neutral pH). To further elaborate on the

importance of charges for the foaming properties of CD and TD re-

lated peptides, the foaming properties of GliaTryp fractions were

studied in the presence of 2.0% (w/v) NaCl.

3.6. Effect of NaCl on the foaming properties of GliaTryp fractions

Before studying the effect of NaCl on the foaming properties of

the GliaTryp fractions, the protein solubility was determined in the

presence of 2.0% (w/v) of it. In general, NaCl addition decreased

peptide solubility, except for GliaTrypsol0-80 at pH 8.0 and 12.0

(Fig. 1B). The solubility of GliaTrypinsol decreased by more than

20% at all pH conditions tested. This was expected as this fraction

contains the largest peptides. While salting out of proteins/pep-

tides is based on differences in hydrophobicity, at increasing ionic

strength, larger peptides tend to precipitate earlier than smaller

peptides (Scopes, 1993). However, gliadin peptides were much less

sensitive to ionic precipitation by NaCl than gliadin itself (Thewis-

sen et al., 2011).

Fig. 2shows the effect of 2.0% (w/v) NaCl on the foaming prop-erties of GliaTryp fractions. In general, FV was slightly higher in the

presence of 2.0% NaCl than when no NaCl was added. The effect of

A B

C D

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0500

2.0 6.7 8.0 12.0

pH

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0500

2.0 6.7 8.0 12.0

pH

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0500

2.0 6.7 8.0 12.0

pH

0.0200

0.0250

0.0300

0.0350

0.0400

0.0450

0.0500

2.0 6.7 8.0 12.0

pH

Surfacetension(Nm-1)




Fig. 3. Surface tension values of GliaTrypsol0-80 (A), GliaTrypsol80-90 (B), GliaTrypsol90+ (C) and GliaTrypinsol (D) solutions at pH 2.0, 6.7, 8.0 and 12.0, set at pH using 1.0 MHCl or NaOH without (black) or in the presence of 2.0% NaCl (transparent). Standard deviations are indicated using error bars.



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2.0% NaCl on FS of GliaTryp fractions varied with pH (Fig. 2). Addi-

tion of salts can either positively (Davis, Foegeding, & Hansen,

2004; Popineau et al., 2002) or negatively (Damodaran, Anand, &

Razumovsky, 1998; Zhu & Damodaran, 1994) affect the foaming

properties of proteins/peptides. The improved FS for CD related

peptide fractions after addition of 2.0% NaCl at both strong acidicand alkaline pH can be explained based on the presence of charges

on the peptide chain. At such pHs, FS was the lowest when no NaCl

was added. Salt counter ions can mask charges on the peptide

chain, which results in decreased repulsion and higher peptide

adsorption at the interface (Dickinson, 1999; Foegeding et al.,

2006).

Fig. 3shows the surface tensions of the GliaTryp fractions in thepresence of 2.0% NaCl. For the CD related peptide fractions, surface

EA

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

B

C

D

F

G

0

20

40

60

80

2.0 6.7 8.0 12.0

Area(%)

pH

H

Fig. 4. Distribution of peptides of GliaTryp sol0-80, GliaTrypsol80-90, GliaTrypsol90+ and GliaTrypinsol in solution (respectively A, B, C and D) and in foam 60.0 min after

whipping (respectively E, F, G, H) at pH 2.0, 6.7, 8.0 and 12.0. The quantities of different peptide fractions are expressed as the percentage area of the respective peptide

fraction to the total area of the RP-HPLC (C18) profile.



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tensions were lower in the presence of 2.0% NaCl. In general, the

reduced surface tensions were in line with the increased FV of

CD related peptide fractions after NaCl addition. There was no link

between the surface tension and FV for the TD related peptides.

Finally, addition of NaCl had no impact on the peptide distribu-

tion of foams and the remaining solution. As in the absence of NaCl,

foams from CD related peptide fractions were more enriched in

hydrophobic peptides than solutions, while, for TD related peptidefractions, only subtle differences were observed (results not

shown).

4. Conclusions

A tryptic gliadin hydrolysate was separated into CD or TD de-

rived peptide fractions and their foaming properties were deter-

mined. FVs of CD related peptide fractions vary little with pH,

whereas FVs of TD related peptide fractions increased from acidic

to alkaline pH. In addition, FS of CD related peptide fractions were

maximal near neutral pH, while those of TD related peptide frac-

tions showed no clear optimal pH for maximal FS. Furthermore,

the more hydrophobic peptides within the CD related peptide frac-

tions seem to mainly contribute to the foaming properties. As the

peptide composition within foams did not vary with pH, the pH

dependent foaming properties of peptides are probably deter-

mined by charges. Moreover, addition of NaCl in most cases im-

proved the foaming properties. Furthermore, our results indicate

that the pH dependent foaming properties of TD related peptide

fractions resemble most those of native gliadin. From this point

of view, we believe that the foaming behaviour of gliadins as a

function of pH is dictated by their TD. Last but not least, the solu-

bility of gliadin peptides, including those with high ionic strength

and their foaming ability potentially make gliadin hydrolysates

attractive ingredients for the production of aerated food systems.

Acknowledgements

This work is a part of the Methusalem programme Food for theFuture at the K.U. Leuven. Kristof Brijs wishes to acknowledge the

Industrial Research Fund (Katholieke Universiteit Leuven, Leuven,

Belgium) for his position as Industrial Research Fund fellow. Inge

Celus wishes to acknowledge the Instituut voor de aanmoediging

van Innovatie door Wetenschap en Technologie in Vlaanderen

(IWT, Brussels, Belgium) for financial support. The authors thank

Ir. J. Callens for her contribution to the foaming experiments and

fruitful discussions during this work. Further, we like to thank

the unit Molecular and Nanomaterials of the Faculty of Science

(K.U. Leuven) for use of the surface tension torsion balance.

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