Food Chemistry 128 (2011) 606612

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

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

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 maximal 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.

Article history: Received 22 September 2010 Received in revised form 10 January 2011 Accepted 1 March 2011 Available online 5 March 2011 Keywords: Foaming properties Gliadin Hydrolysates Peptide fractions

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, Celus, 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; Thewissen et al., 2011). In the previous work (Thewissen et al., 2011), it was reported that gliadin foams at acidic and alkaline pH are selectively enriched in c-gliadins. In contrast, the levels of a- and c-gliadins 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.
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, triuoroacetic acid. Corresponding author. Tel.: +32 0 16321634; fax: +32 0 16321997. E-mail addresses: inge.celus@biw.kuleuven.be (I. Celus), kristof.brijs@biw.kuleuven.be (K. Brijs), jan.delcour@biw.kuleuven.be (J.A. Delcour). 0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2011.03.007

In order to improve the water solubility and foaming properties over a wider pH range, gliadin can be enzymatically hydrolysed. To the best of our knowledge, no research efforts have been made on the foaming properties of gliadin hydrolysates. In contrast, literature reports on the foaming properties of gluten hydrolysate mixtures. Hydrolysis of gluten leads to peptides that either have higher or lower foaming properties than the parent material. Linars, Larr, 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 between 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

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foaming properties of structural different gliadin peptides. The primary 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 to insights 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 Netherlands). Trypsin from porcine pancreas was from SigmaAldrich (Bornem, Belgium). Denatured ethanol (97% v/v) was from Brenntag (Mlheim/Ruhr, Germany). All chemicals, solvents and reagents were purchased from SigmaAldrich and were of analytical grade unless otherwise specied. 2.2. Methods 2.2.1. Gliadin hydrolysis and fractionation of the resulting peptide mixture Tryptic gliadin hydrolysis was performed as described by Thewissen, 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; 50 C; 3.0 h). The pH was kept at pH 8.0 by manual addition of 2.0 M NaOH. 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 freezedried. GliaTrypsol was further fractionated by graded ethanol precipitation as described by Thewissen 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 nal 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; 4 C), suspended in deionised water and freeze-dried. The ethanol concentration in the supernatant was further increased to 90% (v/v) and the precipitated material (GliaTrypsol8090) was recovered as described before. Ethanol was removed from the remaining supernatant (GliaTrypsol90+) by rotary evaporation (50 C). 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 ofcial method 990.03 (AOAC, 1995) to an automated Dumas protein analysis 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 high performance 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 electrochemical detector. During acid hydrolysis, Gln and Asn are transformed 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 compositions of gliadin and GliaTryp fractions were determined by the method of Englyst 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 suspensions, were determined by the above mentioned Dumas method, 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 of Caessens, Gruppen, Visser, van Aken, and Voragen (1997) with small modications. 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 lter (thickness: 5 mm; diameter: 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 dened as the percentage of FV remaining after 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 lter 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 coefcient of variation for the determination of FV and FS was calculated based on a vefold determination with a typical sample and did not exceed 10%. 2.2.4. Surface tension measurements Surface tensions (Nm1) of peptide solutions were determined at Tr using a torsion balance (model OS Balance/Tensiometer, Bidford on Avon, Alcester, Warwickshire, UK) equipped with a 40.0 mm circumference platinum (DuNuoy) ring. Recipients containing the gliadin peptide solutions were cleaned with acetone and air-dried before use. The coefcient of variation for surface tension values was calculated based on a vefold 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), ltered 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, Alltech Associates, Deereld, 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-

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tonitrile (ACN) (solvent B), both containing 0.1% (v/v) triuoroacetic acid (TFA). Peptides were eluted with a linear gradient from 0% to 100% solvent B in 160 min at a ow rate of 0.5 ml/min and detected by absorbance detection at 214 nm. 2.2.6. Size-exclusion HPLC Size-exclusion HPLC (SE-HPLC) was performed with a BiosepSEC-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 ltered through a 0.45 lm PES membrane (Millipore). The injection volume was 60 ll, the ow rate 1.0 ml/min, and the temperature 30 C. 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 gliadin peptide mixture (Thewissen et al., 2010). Based on their AA compositions, GliaTrypsol0-80 and GliaTrypsol80-90 are related to the CD, whereas GliaTrypsol90+ and GliaTrypinsol are related to the gliadin TDs (Thewissen et al., 2010). The highest MW peptides were found in GliaTrypinsol, followed by those in GliaTrypsol0-80, GliaTrypsol80-90 and GliaTrypsol90+ (SE-HPLC results not shown). RP-HPLC analysis showed that GliaTrypsol90+ peptides eluted rst (most hydrophilic conditions), followed by those of GliaTrypsol80-90, GliaTrypsol0-80 and GliaTrypinsol (most hydrophobic conditions), respectively (results not 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 1 lists the results of a partial chemical analysis of the different GliaTryp fractions. Protein yields are dened as the ratio of protein weight of a peptide fraction to the protein weight of GliaTryptotal, and expressed as a percentage. The soluble peptides (GliaTrypsol) represent 86% of GliaTryptotal. After graded ethanol precipitation of GliaTrypsol, almost half of the soluble peptide fraction was recovered in GliaTrypsol0-80 (Table 1). The protein contents of GliaTrypsol0-80 and GliaTrypsol80-90 were very high [99%

and 93% (w/w, db), respectively], while those of GliaTrypsol90+ and GliaTrypinsol were 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 glucose were present in GliaTrypsol90+. Glucose probably originates from dextrins, which are liberated from starch. GliaTrypsol90+ and GliaTrypinsol also 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 solubility 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 GliaTrypinsol was 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 solubility 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 GliaTrypsol80-90) 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 constant, FS of both fractions varied as a function of pH. Maximal FS values for GliaTrypsol0-80 and GliaTrypsol80-90 were obtained at pH 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 native 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 contrast, 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 Gliadin GliaTrypsol GliaTrypsol0-80 GliaTrypsol 80-90 GliaTrypsol 90+ GliaTrypinsol 100 86 46 20 20 14 85 85 99 93 54 56 1.2 n.d. 1.3 1.6 15.1 1.7 5.07 n.d. 1.43 2.84 19.75 1.15 Galactose 1.55 n.d. 0.51 0.80 7.33 6.22 Arabinose 0.13 n.d. 0.12 0.04 0.09 0.12 Xylose 0.05 n.d. 0.07 0.05 0.03 0.00 Mannose 0.45 n.d. 0.17 0.61 0.84 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.

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A 0.40
Solubilised protein (%, w/v)
0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 2.0 6.7 8.0 12.0

pH

B
Solubilised protein (%, w/v)

0.40 0.35 0.30 0.25 0.20 0.15 0.10 0.05 0.00 2.0 6.7 8.0 12.0

3.3.3. TD related peptide fractions FV of TD related peptide fractions (GliaTrypsol90+ and GliaTrypinsol) increased from acidic to alkaline pH (Fig. 2C and D). At each particular pH, FV of GliaTrypsol90+ was lower than for GliaTrypinsol. No residual foam was left 60 min after the start of whipping of GliaTrypsol90+ solution at pH 2.0, while FS was only about 30% at the other pH values. In contrast, the FS of GliaTrypinsol at acidic pH was similar to that at alkaline pH and higher than that of GliaTrypsol90+. The lower FS of GliaTrypsol90+ than that of GliaTrypinsol 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 between 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.

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.

3.4. Surface tension of GliaTryp fractions Due to cohesive (van der Waals) interactions between the molecules at airliquid interfaces, they have a surface tension. Substances 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-active properties of solutions of CD and TD related gliadin peptide

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

A
FV (mL), FS (%)

100 80 60 40 20 0 2.0 6.7 8.0 12.0

B
FV (mL), FS (%)

100 80 60 40 20 0 2.0 6.7 8.0 12.0

pH

pH

C
FV (mL), FS (%)

100 80 60 40 20 0 2.0 6.7 8.0 12.0

D
FV (mL), FS (%)

100 80 60 40 20 0 2.0 6.7 8.0 12.0

pH

pH

Fig. 2. Initial foam volume (FV, solid) and foam stability (FS, transparent) 1.0 h after foam formation of GliaTrypsol0-80 (A), GliaTrypsol8090 (B), GliaTrypsol90+ (C) and GliaTrypinsol (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).

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fractions at different pHs to their corresponding foaming properties, the surface tensions of gliadin peptide solutions were determined (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 constant FVs as function of pH. The surface tension values of TD related peptide solutions (GliaTrypsol90+ and GliaTrypinsol) were lower than those of their CD counterparts (GliaTrypsol0-80 and GliaTrypsol80-90) 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 surface 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, remaining 60 min after the start of whipping, and the remaining solution. It is based on differences in hydrophobicity, although MW also inuences 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), further referred to as RP fractions A, B, C and D, respectively. The percentage 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 hydrophobic 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 the pH, 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 certain 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, peptides carry a net charge which results in electrostatic repulsion between peptides at the interface, so that less peptides adsorb at the interface 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 related peptide fractions (Thewissen et al., 2010), which may indicate 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 related 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/peptides 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 (Thewissen et al., 2011). Fig. 2 shows the effect of 2.0% (w/v) NaCl on the foaming properties 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

Surface tension (Nm-1)

Surface tension (Nm-1)

0.0500 0.0450 0.0400 0.0350 0.0300 0.0250 0.0200 2.0 6.7 8.0 12.0

0.0500 0.0450 0.0400 0.0350 0.0300 0.0250 0.0200 2.0 6.7 8.0 12.0

pH

pH

Surface tension (Nm-1)

0.0500 0.0450 0.0400 0.0350 0.0300 0.0250 0.0200 2.0 6.7 8.0 12.0

Surface tension (Nm-1)

0.0500 0.0450 0.0400 0.0350 0.0300 0.0250 0.0200 2.0 6.7 8.0 12.0

pH
+

pH

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 M HCl or NaOH without (black) or in the presence of 2.0% NaCl (transparent). Standard deviations are indicated using error bars.

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A
Area (%)

80 60

E
Area (%)

80 60 40 20 0

40 20 0 2.0 6.7 8.0 12.0

2.0

6.7

8.0

12.0

pH

pH

B
Area (%)

80 60 40 20 0 2.0 6.7 8.0 12.0

F
Area (%)

80 60 40 20 0 2.0 6.7 8.0 12.0

pH

pH

C
Area (%)

80 60

G
Area (%)

80 60 40 20 0

40 20 0 2.0 6.7 8.0 12.0

2.0

6.7

8.0

12.0

pH

pH

D
Area (%)

80 60

H
Area (%)

80 60 40 20 0

40 20 0 2.0 6.7 8.0 12.0

2.0

6.7

8.0

12.0

pH

pH

Fig. 4. Distribution of peptides of GliaTrypsol0-80, 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) prole.

2.0% NaCl on FS of GliaTryp fractions varied with pH (Fig. 2). Addition 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 acidic and 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. 3 shows the surface tensions of the GliaTryp fractions in the presence of 2.0% NaCl. For the CD related peptide fractions, surface

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B.G. Thewissen et al. / Food Chemistry 128 (2011) 606612 AOAC (1995). Method 990.03. Washington, DC: Association of Ofcial Analytical Chemists. Bks, F., Zawistowska, U., & Bushuk, W. (1983). Protein-lipid complexes in the gliadin fraction. Cereal Chemistry, 60(5), 371378. Brot, S., Popineau, Y., Compoint, J. P., Blassel, C., & Chaufer, B. (2001). Ultraltration to fractionate wheat polypeptides. Journal of Chromatography B, 753(1), 2935. Caessens, P. W. J. R., Gruppen, H., Visser, S., van Aken, G. A., & Voragen, A. G. J. (1997). Plasmin hydrolysis of beta-casein: Foaming and emulsifying properties of the fractionated hydrolysate. Journal of Agricultural and Food Chemistry, 45(8), 29352941. Damodaran, S., Anand, K., & Razumovsky, L. (1998). Competitive adsorption of egg white proteins at the airwater interface. Direct evidence for electrostatic complex formation between lysozyme and other egg proteins at the interface. Journal of Agricultural and Food Chemistry, 46(3), 872876. Davis, J. P., Foegeding, E. A., & Hansen, F. K. (2004). Electrostatic effects on the yield stress of whey protein isolate foams. Colloids and Surfaces B-Biointerfaces, 34(1), 1323. Dickinson, E. (1999). Adsorbed protein layers at uid interfaces: Interactions, structure and surface rheology. Colloids and Surfaces B-Biointerfaces, 15(2), 161176. Drago, S. R., & Gonzalez, R. J. (2001). Foaming properties of enzymatically hydrolysed wheat gluten. Innovative Food Science and Emerging Technologies, 1, 269273. Englyst, H. N., & Cummings, J. H. (1984). Simplied method for the measurement of total non-starch polysaccharides by gasliquid-chromatography of constituent sugars as alditol acetates. Analyst, 109(7), 937942. Foegeding, E. A., Luck, P. J., & Davis, J. P. (2006). Factors determining the physical properties of protein foams. Food Hydrocolloids, 20(23), 284292. Kong, X. Z., Zhou, H. M., & Qian, H. F. (2007). Enzymatic preparation and functional properties of wheat gluten hydrolysates. Food Chemistry, 101(2), 615620. Linars, E., Larr, C., Lemeste, M., & Popineau, Y. (2000). Emulsifying and foaming properties of gluten hydrolysates with an increasing degree of hydrolysis: Role of soluble and insoluble fractions. Cereal Chemistry, 77(4), 414420. Mita, T., Ishida, E., & Matsumoto, H. (1978). Physicochemical studies on wheatprotein foams. 2 relationship between bubble-size and stability of foams prepared with gluten and gluten components. Journal of Colloid and Interface Science, 64(1), 143153. Patino, J. M. R., Sanchez, C. C., & Nino, M. R. R. (2008). Implications of interfacial characteristics of food foaming agents in foam formulations. Advances in Colloid and Interface Science, 140(2), 95113. Popineau, Y., Huchet, B., Larr, C., & Brot, S. (2002). Foaming and emulsifying properties of fractions of gluten peptides obtained by limited enzymatic hydrolysis and ultraltration. Journal of Cereal Science, 35(3), 327335. Rombouts, I., Lamberts, L., Celus, I., Lagrain, B., Brijs, K., & Delcour, J. A. (2009). Wheat gluten amino acid composition analysis by high-performance anionexchange chromatography with integrated pulsed amperometric detection. Journal of Chromatography A, 1216(29), 55575562. Scopes, R. K. (1993). Protein purication, principles and practice. New York, USA: Springer-Verlag. Shewry, P. R., & Tatham, A. S. (1990). The prolamin storage proteins of cereal seeds structure and evolution. Biochemical Journal, 267(1), 112. Thewissen, B. G., Celus, I., Brijs, K., & Delcour, J. A. (2011). Foaming properties of wheat gliadin. Journal of Agricultural and Food Chemistry, 59, 13701375. Thewissen, B. G., Celus, I., Brijs, K., & Delcour, J. A. (2010). Fractionation of tryptic gliadin hydrolysates based on proline levels. Journal of Cereal Science, 52, 275281. Uthayakumaran, S., Tomoskozi, S., Tatham, A. S., Savage, A. W. J., Gianibelli, M. C., Stoddard, F. L., et al. (2001). Effects of gliadin fractions on functional properties of wheat dough depending on molecular size and hydrophobicity. Cereal Chemistry, 78(2), 138141. Wang, J. S., Zhao, M. M., Bao, Y., Hong, T., & Rosella, C. M. (2008). Preparation and characterisation of modied wheat gluten by enzymatic hydrolysisultraltration. Journal of Food Biochemistry, 32(3), 316334. Zhu, H. M., & Damodaran, S. (1994). Proteose peptones and physical factors affect foaming properties of whey-protein isolate. Journal of Food Science, 59(3), 554560.

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 distribution 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 peptide fractions, only subtle differences were observed (results not shown). 4. Conclusions A tryptic gliadin hydrolysate was separated into CD or TD derived peptide fractions and their foaming properties were determined. 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 fractions showed no clear optimal pH for maximal FS. Furthermore, the more hydrophobic peptides within the CD related peptide fractions 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 determined by charges. Moreover, addition of NaCl in most cases improved 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 solubility 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 the Future 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 nancial 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. References
AACC (2000). Approved methods of the aacc. St. Paul, MN, USA: American Association of Cereal Chemists Inc.