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8/12/2019 Foaming Properties of Tryptic Gliadin
1/7
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
j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / f o o d c h e m
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.2011.03.0078/12/2019 Foaming Properties of Tryptic Gliadin
2/7
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
8/12/2019 Foaming Properties of Tryptic Gliadin
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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).
B.G. Thewissen et al./ Food Chemistry 128 (2011) 606612 609
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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)
Surfacetension(Nm-1)
Surfacetension(Nm-1)
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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