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Article
Exploring the relationship between structural and airwater interfacial properties of wheat (Triticum aestivum L.) gluten hydrolysates in a food system relevant pH range Arno G.B. Wouters, Ellen Fierens, Ine Rombouts, Kristof Brijs, Iris J. Joye, and Jan A. Delcour J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05062 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on January 28, 2017
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Journal of Agricultural and Food Chemistry
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Exploring the relationship between structural and air-water interfacial
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properties of wheat (Triticum aestivum L.) gluten hydrolysates in a
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food system relevant pH range
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Arno G.B. Woutersa*, Ellen Fierensa, Ine Romboutsa, Kristof Brijsa, Iris J. Joyea,b Jan A. Delcoura
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a
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Research Center (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, B-3001 Leuven, Belgium.
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b
10
Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition
Food Science Department, University of Guelph, 50 Stone Road East Guelph, Ontario, N1G 2W1,
Canada.
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*Corresponding author.
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Tel.: +32 (0) 16 372035
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E-mail address:
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT
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The relationship between structural and foaming properties of two tryptic and two peptic wheat
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gluten hydrolysates was studied at different pH conditions. The impact of pH on foam stability
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(FS) of the samples heavily depended on the peptidase used and the degree of hydrolysis
19
reached. Surface dilatational moduli were in most, but not all, instances related to FS, implying
20
that, while the formation of a visco-elastic protein hydrolysate film is certainly important, this is
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not the only phenomenon which determines FS. In contrast to what might be expected, surface
22
charge was not a major factor contributing to FS, except when close to the point-of-zero-charge.
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Surface hydrophobicity and intrinsic fluorescence measurements suggested that changes in
24
protein conformation take place when the pH is varied, which can in turn influence foaming.
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Finally, hydrolyzed gluten proteins formed relatively large particles, suggesting that protein
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hydrolysate aggregation probably influences its foaming properties.
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Keywords: Gluten hydrolysates, pH, foam, air-water interface, protein conformation, interfacial
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properties
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Journal of Agricultural and Food Chemistry
1. INTRODUCTION
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A foam is a dispersion of a gaseous phase in a liquid. Its creation requires energy input is. Foams
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destabilize rapidly as they are thermodynamically unstable 1. Both foaming capacity (FC) and
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foam stability (FS) are important foam properties which can be influenced by the presence of
34
low molecular weight surfactants or proteins 2. Low molecular weight surfactants and proteins
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tend to diffuse to and adsorb at an air-water (A-W) interface. Once adsorbed, they stabilize the
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interface in different ways1-3:
37
-
tension,
38 39
-
-
proteins can sterically hinder gas bubbles from approaching each other and merging, and
42 43
proteins and ionic surfactants promote electrostatic repulsion between the gas bubbles which they coat,
40 41
proteins and low molecular weight surfactants at the interface lower the surface
-
proteins likely undergo changes in conformation upon adsorption at the A-W interface.
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They expose their more hydrophobic regions towards the air phase and form a
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viscoelastic film around the gas cells through protein – protein interactions which
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stabilize the A-W interface. While such interactions can be hydrogen bonds or of the van
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der Waals type, especially electrostatic and hydrophobic interactions are considered to
48
be important in this context 1, 2, 4.
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Not all (plant) proteins are evenly suited as foaming agents. For example, wheat (Triticum
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aestivum L.) gluten protein, a co-product of the industrial starch isolation, has very low solubility
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in aqueous media and very low ability to form and stabilize foams 5-7. Enzymatic hydrolysis not 3 ACS Paragon Plus Environment
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only strongly increases the solubility of gluten but also improves its foaming properties 8. Such
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enzymatic hydrolysis induces three major structural changes in proteins: a decrease in average
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molecular mass, a better accessibility of hydrophobic regions and a higher level of ionizable
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groups 9. These structural changes affect intermolecular interactions between the peptides in a
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hydrolysate and consequently their foaming and interfacial behavior.
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Also, the structure of peptides as well as inter-peptide interactions at an A-W interface are
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strongly influenced by environmental conditions such as pH. The effect of pH on plant protein
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hydrolysate foaming properties has been addressed by some researchers. The FC of pea protein
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isolates hydrolyzed with papain is lower at pH 8.0 than at pH 3.0, 5.0 and 7.0
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observation was made for peanut protein isolates hydrolyzed with Alcalase, which not only have
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good FC but also high FS at pH 3.0
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with subtilisin) increases with pH (up to pH 8.0), after which it decreases again (at pH 11.0) 12.
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For gluten protein chymotrypsin hydrolysates, Popineau et al.
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properties (FC and FS) at pH 4.0 than at pH 6.5, while Drago & González 14 observed an opposite
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trend for such hydrolysates when obtained using a fungal protease. They reported better
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foaming properties at pH 6.5 and pH 9.0 than at pH 4.0. Another study noted no differences in
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foaming capacity at pH 5.0, 7.0 and 8.0 of papain gluten hydrolysates 15. The impact of pH on
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foaming of wheat gluten hydrolysates is thus complex and depends on the peptidase used and
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the hydrolysis conditions. The latter determine which peptides are solubilized, the properties of
71
which (including their charge, conformation and aggregation propensity) depend on conditions
72
such as pH and temperature. For non-hydrolyzed proteins extracted from e.g. hemp seed
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lentil
17
and soy
18
11
10
. A similar
. In contrast, FS of barley protein hydrolysates (produced
13
reported better foaming
16
,
, pH-induced structural changes and their effect on foaming have been
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described. For plant protein hydrolysates, similar studies relating pH to structural changes are
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rare. Some authors, including some of the ones mentioned above 10-12, 14 have speculated on the
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molecular phenomena taking place at the A-W interface. However, it has often only been
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assumed but not experimentally shown [for example by measuring zeta potential (ZP)], that the
78
surface charge of proteins and peptides plays a key role in pH-dependent foaming
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example, Thewissen et al.
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from which they concluded this had to be related to the surface charge of the peptides. In
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addition to the above, pH-induced conformational changes in hydrolyzed plant protein which
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affect A-W interfacial properties remain to be investigated.
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This background shows that in order to better understand the structure – function relationship
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of wheat gluten hydrolysates, a comprehensive study is necessary. Here, foaming, A-W
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interfacial (decrease of surface tension and surface dilatational moduli) and structural (surface
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charge, protein conformation and aggregation) properties of wheat gluten hydrolysates were
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evaluated at various pH values. The work reported has increased our understanding of how
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hydrolyzed proteins at A-W interfaces impact FS.
19
10, 14
. For
observed pH and salt dependent foaming of gliadin hydrolysates
89 90 91
2. MATERIALS AND METHODS 2.1 Materials
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Commercial wheat gluten was kindly provided by Tereos Syral (Aalst, Belgium). It contained
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82.4% protein (N x 5.7) on dry matter basis when determined using an adaptation of AOAC
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Official Method 990.03
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Waltham, MA, USA). Trypsin (EC 3.4.21.4) from porcine pancreas (reference from the distributor
20
to an EA1108 Elemental Analyzer (Carlo Erba/Thermo Scientific,
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T0303) and pepsin (EC 3.4.23.1) from porcine gastric mucosa (reference from the distributor
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P6887) were purchased from Sigma-Aldrich (Bornem, Belgium), as were all other chemicals,
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solvents and reagents.
99 100
2.2 Enzymatic hydrolysis
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A 6.0% (wprotein/v) wheat gluten aqueous dispersion was incubated with trypsin or pepsin at pH-
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stat conditions in a Titrino 718 device (Metrohm, Herisau, Switzerland). For each enzyme, gluten
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was hydrolyzed to degrees of hydrolysis (DH) 2 and 6. The DH reflects the percentage of initially
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present peptide bonds which have been hydrolysed (see below). For tryptic hydrolysis, pH-stat
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conditions were 50 °C, pH 8.0 and an enzyme to substrate ratio of 1:480 (DH 2) or 1:20 (DH 6)
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on protein mass basis was used. For peptic hydrolysis, the reactions were carried out at 37 °C,
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pH 3.5 and an enzyme to substrate ratio of 1:1200 (DH 2) or 1:300 (DH 6) on protein mass basis
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was used. When the desired DH was reached, the pH was adjusted to 6.0 and proteolysis was
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stopped by heating the protein suspension for 15 min at 95 °C. It should be noted that the
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heating procedure may cause limited peptide aggregation. This will be further discussed in
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section 3.6. The mixtures were then centrifuged (10 min, 12,000 g) at room temperature, and
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the supernatants filtered and then freeze-dried. All further analyses, including those of protein
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contents (carried out as outlined in Section 2.1), were conducted on the freeze-dried
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supernatants of DH 2 or DH 6 tryptic (further referred to as T2 and T6, respectively) and peptic
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(further referred to as P2 and P6, respectively) hydrolysates.
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2.3 Determination of degree of hydrolysis
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DH is the percentage of peptide bonds hydrolyzed (h) relative to the total number of peptide
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bonds (htot) per unit weight present in wheat gluten protein. It was calculated from the quantity
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of NaOH (trypsin) or HCl (pepsin) solution used to keep the pH constant during hydrolysis:
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% =
. .
=
. .
(1)
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With X the consumption (mL) of NaOH or HCl solution needed to keep the pH during hydrolysis
123
constant and Mx the molarity of the acid or base (respectively 0.50 and 0.20 M). The term α is a
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measure for the degree of dissociation of the α-NH3+ (neutral or alkaline conditions) or α-COOH
125
group (acidic conditions). Under the given conditions, for tryptic hydrolysis α is 0.89 21, whereas
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for peptic hydrolysis it is 0.29
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[milli-equivalents (meqv)/g protein] and htot is the theoretical number of peptide bonds per unit
128
weight present in gluten protein. Nielsen, Petersen and Dambmann 23 calculated the latter to be
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8.3 meqv/g protein.
22
. Mp is the mass of protein used, h are hydrolysis equivalents
130 131
2.4 Measurement of protein solubility
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First, solutions of T2, T6, P2 and P6 [0.075% (wprotein/v)] in 0.050 M sodium phosphate buffer (pH
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6.8) containing 2.0% of sodium dodecyl sulfate (SDS) were made. Under such conditions, the
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samples were completely soluble and are further referred to as reference samples. Then, T2, T6,
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P2 and P6 [0.15% (wprotein/v)] were suspended in deionized water and the pH was adjusted to pH
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3.0, 5.0 and 7.0 by adding small amounts of 1.0 M NaOH or 1.0 M HCl. The samples were
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centrifuged (10 min, 10,000 g) to remove any insoluble material. An aliquot of the obtained
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supernatants (400 µL) was diluted with an extra 400 µL 0.100 M sodium phosphate buffer (pH
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6.8) containing 4.0% SDS. An aliquot (40 µL) of these diluted samples was loaded on a BioSep 7 ACS Paragon Plus Environment
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SEC-S3000 column (Phenomenex, Torrance, CA, USA) and analyzed using a Shimadzu (Kyoto,
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Japan) LC-2010 integrated HPLC system with peptide elution monitoring at 214 nm. Samples
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eluted at a flow rate of 0.50 mL/min using 0.050 M sodium phosphate buffer (pH 6.8) containing
143
2.0% SDS at 30 °C. Peptide solubility was determined by calculating the area in the
144
chromatographic profiles and expressing it relative to the area in the profile of a reference
145
sample (100% soluble).
146 147
2.5 Analysis of foaming properties
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Foaming properties were determined with a standardized whipping test based on Caessens et
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al. 24. Solutions of T2, T6, P2 and P6 [0.050% (wprotein/v)] were prepared and the pH was adjusted
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to pH 3.0, 5.0 and 7.0 as above. An aliquot (50.0 mL) of these solutions was placed in a
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graduated glass cylinder (internal diameter 60.0 mm) in a water bath at 20 °C. After
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temperature equilibration for 15 min, it was whipped for 70 s using a rotating propeller (outer
153
diameter 45.0 mm, thickness 0.4 mm) at 2,000 rpm. After whipping, the propeller was
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immediately removed and the glass cylinder sealed with Parafilm M (Bemis, Neenah, WI, USA)
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to avoid foam disruption by air circulation. The FC was the foam volume exactly 2 min after the
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start of whipping. The foam volume was then also measured at 4, 10, 15, 30, 45 and 60 min
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after the start of whipping. The decrease of foam volume over time was an indication for the FS
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of a given sample. Based on the foam height and the cylinder internal diameter, foam volume
159
was calculated and expressed in mL.
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2.6 (Oscillating) pendant drop measurements
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Solutions [0.050% (wprotein/v)] of T2, T6, P2 and P6 in deionized water, adjusted to pH 3.0, 5.0
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and 7.0 as above, were introduced in a Theta optical tensiometer (Biolin Scientific Attension,
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Stockholm, Sweden) to create a pendant drop with a fixed volume of 8 µL. For every drop, the
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decrease in surface tension was measured over a 10 min time interval to assess protein
166
adsorption and rearrangement at the A-W interface. In doing so, images were taken (1 frame
167
per 7 seconds). A sinusoidal oscillation (50 cycles) was then performed at a frequency of 1 Hz
168
with an amplitude set at 1.00 in the OneAttension software (Biolin Scientific Attension), which
169
corresponded to a volume of ± 1 µL. During oscillation, images were recorded (7 frames per
170
second). The surface dilatational elastic modulus E’ of the interfacial film could be calculated
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from the drop shape analysis data during oscillation. Measurements were conducted at room
172
temperature (22 ± 2 °C). After each measurement, the device was thoroughly cleaned and the
173
surface tension of pure water was checked to be 72.0 ± 0.5 mN/m, before initiating the next
174
measurement.
175
2.7 Measurements of zeta potential
176 177
Solutions of T2, P2, T6 and P6 [0.15% (wprotein/v)] in deionized water, adjusted to pH 3.0, 4.0, 5.0,
178
6.0 and 7.0 as above, were transferred to a disposable capillary zeta cell (Malvern Instruments,
179
Malvern, United Kingdom) to determine the ZP in a Zetasizer Nano ZS (Malvern) based on laser
180
Doppler micro-electrophoresis. Measurements were conducted at room temperature (22 ± 2
181
°C).
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2.8 Determination of surface hydrophobicity
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The surface hydrophobicity of all samples was determined with 1-anilino-8-naphtalene sulfonic
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acid (ANS) as fluorescent probe. Samples were dissolved in deionized water and the pH adjusted
186
to pH 3.0, 4.0, 5.0, 6.0 and 7.0 as above. They were then diluted with deionized water (adjusted
187
to the same pH) to protein concentrations of 0.18, 0.36, 0.54, 0.72 and 0.90 mg/mL. Of each
188
diluted sample, 200.0 µL was transferred to a 96-well plate in duplicate, and 10.0 µL ANS
189
solution (8.0 mM in deionized water) was added. The fluorescence emission intensity of the
190
protein samples was recorded at 480 nm with a Synergy Multi-Mode Microplate Reader (BioTek,
191
Winooski, VT, USA) after excitation at 390 nm. The relative fluorescence intensity was calculated
192
as the intensity of the fluorescence of the protein-ANS mixture minus that of the control sample
193
(ANS with water) fluorescence, which was then divided by the fluorescence intensity of the
194
control sample. The slope of the plot of relative fluorescence intensity as a function of protein
195
concentration for each sample represents the surface hydrophobicity. Measurements were
196
conducted at room temperature (22 ± 2 °C).
197 198
2.9 Analysis of intrinsic tryptophan fluorescence
199
Changes in the local environment of tryptophan residues in a protein chain can be assessed
200
from the fluorescence spectrum of the tryptophan indole ring. The intrinsic tryptophan
201
fluorescence of T2, T6, P2 and P6 solutions [0.010% (wprotein/v) in deionized water] adjusted to
202
pH 3.0, 5.0 and 7.0 as above was evaluated with a Fluoromax 4 fluorospectrometer (Horiba
203
Jobin Yvon, Edison, NJ, USA). The excitation wavelength was 295 nm with a 2 nm slit width,
204
while the emission spectrum was recorded from 300 to 450 nm using a 3 nm slit width.
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Measurements were conducted at room temperature (22 ± 2 °C). 10 ACS Paragon Plus Environment
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2.10 Dynamic light scattering
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Solutions of T2, P2, T6 and P6 [0.15% (wprotein/v) in deionized water] adjusted to pH 3.0, 5.0 and
209
7.0 as above were placed in disposable cuvettes to measure dynamic light scattering in a
210
Zetasizer Nano ZS (Malvern). The particle size distribution (PSD) was calculated from this
211
scattering pattern with the Stokes-Einstein relationship using the Malvern Zetasizer software.
212
Measurements were conducted at room temperature (22 ± 2 °C).
213 214
2.11 Statistical analysis
215
Protein solubility of the samples at different pH values was determined in duplicate. All foam
216
related experiments, ZP, dynamic light scattering, surface hydrophobicity, pendant drop and
217
intrinsic tryptophan fluorescence measurements were carried out at least in fourfold. All data
218
were analyzed using statistical software JMP Pro 11 (SAS Institute, Cary, NC, USA), with a
219
Student t test at a significance level α = 0.05.
220 221 222
3. RESULTS AND DISCUSSION 3.1 Protein solubility
223
The solubility of the peptides ranged from 94 to 100% (Table 1). No impact of pH on solubility
224
was noted for any of the samples (Table 1, capital letters) except for P2 which had a marginally
225
lower solubility at pH 5.0 and pH 7.0 than at pH 3.0. In addition, at pH 5.0 there were no
226
significant (P < 0.05) differences in solubility between the four different samples. At pH 7.0, P6 11 ACS Paragon Plus Environment
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had slightly lower protein solubility than the other samples while at pH 3.0, it was slightly less
228
soluble than P2. Thus, none of the differences observed in foaming or structural properties
229
described in the following paragraphs can be attributed to differences in peptide solubility. The
230
reason that only minor differences were observed is that all insoluble material had already been
231
removed directly after hydrolysis, while in most other studies on the impact of pH on plant
232
protein hydrolysates (see introduction) this was not the case 10, 12-15. We believe that the merit
233
of our approach is that it allows drawing conclusions about the relationship between gluten
234
hydrolysate structural properties and their foaming, thereby surpassing mere differences in
235
levels of solubilized protein.
236 237
3.2 Foaming properties
238
Foaming properties of the four hydrolysates were impacted differently by pH (Figure 1). At a
239
given pH value, the FC did not differ much between different samples (Figure 1). The FC of all
240
samples was significantly (P < 0.05) higher at pH 7.0 than at pH 3.0 or pH 5.0, except for T2,
241
which had higher FC at pH 7.0 than at pH 3.0 but not than that at pH 5.0. Overall, these
242
differences were minor. In terms of FS, all samples sustained at least some foam after 60 min at
243
pH 7.0. However, DH 2 hydrolysates clearly had better FS than DH 6 hydrolysates. At pH 5.0,
244
foams from all hydrolysates destabilized very quickly as illustrated by the steep, fast decrease in
245
foam volume. At pH 3.0, tryptic and peptic hydrolysates clearly behaved differently. While both
246
foams of peptic hydrolysates very rapidly collapsed at pH 3.0, those of tryptic hydrolysates did
247
not. The foam of T2 at pH 3.0 was the most stable of all foam samples produced. Although the
248
foam volume of T6 at pH 3.0 after 60 min was zero, it did not rapidly lose stability the way
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foams of P2 and P6 did. Thus, a pH decrease from 7.0 to 5.0 led to a steep decrease in FS for all
250
hydrolysates. A further pH drop from 5.0 to 3.0 improved FS of tryptic but not peptic
251
hydrolysates. These differences in FS, not only for different samples at a given pH, but also for a
252
given sample at different pH values, might be attributed to the nature of protein films formed at
253
the interface at different pH values. Electrostatic and hydrophobic interactions, because of
254
differences in charge of ionizable groups and peptide conformation at varying pH, probably play
255
a role in this context. All these hypotheses will be explored in the following paragraphs.
256 257
3.2 Air-water interfacial properties
258
The foaming of protein hydrolysates depends on their adsorption at the A-W interface and the
259
nature of the film they there form. Differences in pH values may alter the surface-activity and
260
the ability of gluten hydrolysates to form a strong viscoelastic protein film. Figure 2 shows the
261
decrease of surface tension over time after formation of a pendant drop. The rate and extent of
262
this decrease are indicative for the rates of diffusion and adsorption of the hydrolysate
263
constituents at the interface, as well as for their re-arrangement at the A-W interface, thereby
264
orienting their more hydrophobic regions towards the air phase. For both T2 and T6, the
265
decrease in surface tension was similar at pH 3.0, 5.0 and 7.0 (Figure 2), so the rate and ability
266
of their peptides to diffuse to and continuously adsorb at the A-W interface remained
267
unchanged. Proteins continue to adsorb and re-arrange at the interface until an equilibrium is
268
reached. Data was collected up to 10 min after drop formation. At this time, the surface tension
269
approached a plateau, and it was possible to estimate a value representative for the equilibrium
270
surface tension (Figure 2). To this end, surface tension versus 1/√t (t = Wme) plots (not shown 13 ACS Paragon Plus Environment
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here) were made using the data in Figure 2. From a certain time onwards (and thus at low 1/√t
272
values) linear relationships were observed, which were used to make extrapolations to 1/√t = 0
273
(which accords to t ∞). The laZer yielded surface tension values, indicaWve for equilibrium
274
surface tensions (Table 2). Even though there were some significant differences in these
275
estimated equilibrium surface tension values of either T2 or T6 at different pH values (Table 2,
276
capital letters), these differences were minor. In contrast, peptic hydrolysates were much less
277
able to decrease surface tension at pH 3.0 than at pH 5.0 and 7.0. Also, estimated equilibrium
278
surface tension values for P2 and P6 were much higher at pH 3.0 than at pH 5.0 or pH 7.0 (Table
279
2, capital letters), suggesting that the hydrolysates had a lower affinity for the A-W interface or
280
that they were not able to arrange themselves very efficiently at the A-W interface at this lower
281
pH value. These observations are in line with the fact that both tryptic hydrolysates led to a
282
decent FS at pH 3.0, while peptic hydrolysates did not. After subsequent sinusoidal volume
283
oscillations of the hanging drops, surface dilatational moduli could be determined (Figure 2). E’,
284
which is indicative for the elasticity of the film at the interface, is often related to foam stability,
285
in the sense that elevated film elasticities in many instances are responsible for high stability in
286
protein foams 3, 25. T2 and to a lesser extent T6 had very high E’ at pH 3.0. As expected from the
287
low FS of peptic hydrolysates at pH 3.0, P2 and P6 had very low E’ at pH 3.0. With increasing pH,
288
E’ of both tryptic hydrolysates gradually decreased, while the opposite was observed for both
289
peptic hydrolysates. These had the highest E’ values at pH 7.0. As already stated above, foams
290
of both DH 2 hydrolysates at pH 7.0 were more stable than those of both DH 6 hydrolysates.
291
This is in accordance with the fact that T2 and P2 had significantly (P < 0.05) higher E’ than T6
292
and P6 at pH 7.0. However, not all observations regarding FS were fully in line with E’ values. For
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example, all samples had relatively high E’ at pH 5.0, even though their foams rapidly lost
294
stability at this pH. This suggests that the elasticity obtained through oscillating drop
295
measurements of the protein film in some but not all, and especially not at pH 5.0, cases can
296
explain differences in FS.
297 298
3.4 Surface charge
299
As pH evidently impacts the charge of the ionizable groups in the hydrolysates and thereby may
300
alter their ability to interact at the A-W interface and to stabilize the foam, it may well be at the
301
basis of differences in foaming. At pH 7.0, all samples had comparable ZPs (P > 0.05), with values
302
ranging from -2 mV to -4 mV, except for T6, which had a much lower ZP (-16 mV) (Figure 3). This
303
observation cannot directly be related to differences in foaming properties. Indeed, while P6
304
and T6 had similar low FS at pH 7.0, the former had a much higher ZP than the latter. In
305
addition, these two DH 6 hydrolysates had comparable surface tension (Table 2) and surface
306
dilatational moduli (Figure 2) at pH 7.0. As expected, for all hydrolysates, the zeta potential
307
increased with decreasing pH, as the ionizable groups were gradually protonated. The point-of-
308
zero-charge (PZC), i.e. the pH at which a sample has a ZP of zero, of T2, P2 and P6 was about 5.0,
309
while that recorded for T6 was about 4.0. At this pH, electrostatic repulsion between peptide
310
chains is minimal, so aggregation - with or without loss of solubility - is most probable. These
311
observations are largely in line with the very low FS at pH 5.0 of all samples. However, as
312
discussed in section 3.1, there was no decrease in protein solubility at pH 5.0. It is indeed
313
possible that aggregation occurs at the PZC but apparently only to such extent that it does not
314
impact peptide solubility. Furthermore, in spite of the low FS readings at pH 5.0, all hydrolysates 15 ACS Paragon Plus Environment
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315
still had relatively high E’ values. Thus, the lack of an overall net charge did not prevent the
316
hydrolysates from interacting and forming rather strong protein films. At pH 3.0, while there
317
were some statistically significant (P < 0.05) differences in ZP between some of the samples,
318
these should not be over-interpreted as all ZP values were rather close to zero and differences
319
were only minor. In contrast, there were large differences in FS as well as E’ values between
320
tryptic and peptic hydrolysates at pH 3.0. Thus, the assumption that the pH dependence of
321
foaming is dictated by the charge of ionizable groups in the hydrolysates does not hold true in
322
all cases. Only at the PZC, where FS is very low for all samples, surface charge seems to be
323
directly related to differences in foaming. We hypothesize that the low FS observed at pH 5.0
324
(and thus close to the PZC) is likely related to a lack of electrostatic repulsion between different
325
gas bubbles, which makes their coalescence more likely, rather than to a weakening of the
326
protein films around the gas bubbles. This would mean that at pH 3.0 and 7.0, repulsion
327
(because of the overall positive or negative charge of the protein films) occurs to such extent
328
that it prevents gas bubbles from easily merging. At these pH values, the strength and integrity
329
of the protein films (see section 3.3) probably determine the stability of gluten hydrolysate
330
foams.
331
3.5 Surface hydrophobicity and intrinsic tryptophan fluorescence
332
As pH changes, not only the charge of the peptides varies, but also their conformation may be
333
affected. For various native plant proteins, pH-induced conformational changes have been
334
studied
335
case, irrespective of pH, peptic hydrolysates had a higher surface hydrophobicity than tryptic
336
hydrolysates, and DH 2 hydrolysates had a higher surface hydrophobicity than their DH 6
16-18
, but this has not yet been reported for plant protein hydrolysates. In the present
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337
counterparts (Figure 4). These results are in accordance with RP-HPLC evidence from our team
338
26
339
surface hydrophobicity was observed at pH 3.0 for all samples. Compared to the surface
340
hydrophobicity at pH 4.0, an 8-fold increase of surface hydrophobicity was observed for T2 at
341
pH 3.0, followed by P6 (5.5-fold increase), P2 (5-fold increase) and finally T6 (2-fold increase).
342
These surface hydrophobicity readings (Figure 4) at pH 3.0 seem to suggest that hydrophobic
343
regions are more accessible by ANS at this pH value, possibly indicative for strong differences in
344
conformation. With regard to the surface hydrophobicity readings, it should be mentioned that
345
due to the negative charge on the sulfonic acid group of ANS, electrostatic interactions between
346
proteins and ANS may influence surface hydrophobicity measurements
347
transition from pH 4.0 to pH 3.0, there are barely any functional groups that become positively
348
charged, there is an increase in net positive charge of the peptide mixture due to the
349
protonation of negatively charged carboxylic acid groups. However, while the carboxylic groups
350
of the side chains of glutamic acid and aspartic acid have pKa values of around 4.0, the
351
carboxylic groups originating from the backbone of the protein chain have a pKa of around 2.0
352
and for a significant part will not occur in their protonated form at pH 3.0 29. Additionally, if this
353
were the sole factor involved, it would have been expected that all hydrolysates would have had
354
a similar response, as they had comparable ZPs at pH 3.0. Clearly, this is not the case. Thus,
355
there are some arguments which suggest that the peptides in the hydrolysates might in fact
356
undergo pH-induced conformational changes. These could then contribute to the pH-
357
dependence of their foaming, especially since the surface hydrophobicity increased drastically
358
at pH 3.0, which is the pH where tryptic and peptic hydrolysates had distinctly different foam
that at pH 6.4, P2 and P6 have higher overall hydrophobicity than T2 and T6. The highest
27, 28
. While, upon the
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359
stabilities. However, as similar ZP values (and therefore net surface charges) don’t always
360
correspond to equal levels of ionizable groups, such possible interference by electrostatic
361
interactions between ANS and the peptides at pH 3.0 could not be completely ruled out.
362
Therefore, changes in protein conformation were also studied by analysis of tryptophan
363
fluorescence. Changes in the local environment of this amino acid affect both the wavelength at
364
which the emission intensity is highest (i.e. the maximum emission wavelength) as well as the
365
fluorescence emission spectrum intensity. Shifts in maximum emission wavelength usually
366
indicate changes in polarity of the environment of the amino acid, while changes in intensity are
367
often related to a higher mobility and accessibility of tryptophan for quenchers. Thus, both
368
phenomena report on changes in protein conformation 30. Figure 5 shows the impact of pH on
369
the emission spectra (300-450 nm) of all four hydrolysates. There were notable shifts in the
370
maximum emission wavelengths neither for different samples at a given pH nor within one
371
sample at different pH values. Values of maximum emission wavelengths all ranged between
372
355 and 359 nm, which is a value typical for tryptophan residues which are completely exposed
373
to a polar environment. At the wavelength of maximum emission and at any given pH value, P2
374
had the highest fluorescence intensity, followed by T2, T6 and P6, respectively (all differences
375
were significant, P < 0.05). Additionally, for any given sample, the fluorescence emission
376
intensities at pH 7.0 were significantly (P < 0.05) higher than at pH 5.0, which in turn were
377
significantly higher than the values recorded at pH 3.0. All this confirmed that it is likely that
378
changing the pH induces changes in the conformation of the proteins. Furthermore, as all
379
samples had a similar change in fluorescence emission intensity, this and the surface
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380
hydrophobicity values at different pH values did not explain why tryptic hydrolysates had
381
relatively high FS, while peptic hydrolysates had very low FS at pH 3.0.
382 383
3.6 Particle size distribution
384
In the previous paragraphs, it was suggested that the peptides may occur as aggregates. Even
385
when it would not affect the overall peptide solubility, such aggregation may heavily influence
386
the A-W interfacial properties of gluten hydrolysates. PSDs were measured with dynamic light
387
scattering. There was quite some variation between different measurements of the same
388
sample at a given pH. This may be due to the fact that aggregates present in such hydrolysates
389
are loosely assembled, and re-organize rather easily upon changes in the environment. The
390
PSDs shown in Figure 6 are averages of at least 12 repeated measurements, and are indicative
391
of the aggregation of different samples under these conditions. In all cases, samples contained
392
particles ranging from 10 up to 4,000 nm in size (Figure 6). Thus, enzymatic hydrolysis probably
393
yielded peptides which have a tendency to form (soluble) aggregates. It should be mentioned
394
that larger particles scatter more light meaning that their relative abundance based on the PSDs
395
in Figure 6 should be interpreted with care. PSDs of P2 and P6 were not or only slightly affected
396
by varying the pH. It seemed that, although intrinsic fluorescence and, even more so, surface
397
hydrophobicity measurements indicated that conformational changes occurred in P2 and P6,
398
these changes did not impact their aggregation, as measured by dynamic light scattering. In
399
contrast, the impact of pH on the PSDs of tryptic hydrolysates was much more pronounced. T2
400
had polydisperse and rather similar PSDs at pH 3.0 and 7.0. While at pH 7.0 T2 and T6 both had
401
polydisperse PSDs, the latter had a higher average particle size. At pH 3.0, a monodisperse peak 19 ACS Paragon Plus Environment
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402
of particles at around 530 nm was observed for T6. At pH 5.0, all samples had rather
403
monodisperse PSDs. One could hypothesize that the aggregates at pH 5.0 would be larger than
404
those at other pH values because of the proximity to the PZC, where no electrostatic repulsion
405
occurs, thereby leading to formation of an impaired protein film at the A-W interface. However,
406
this was not always the case. For example, T6 solutions contained even larger (> 1,000 nm)
407
particles at pH 3.0 than at pH 5.0, but led to better FS at pH 3.0 than at pH 5.0. It is difficult to
408
assess how the presence of these aggregates affects the A-W interfacial and foaming properties
409
of gluten hydrolysates. Aggregation may slow down the diffusion to and adsorption at the
410
interface but may also affect the ability of the hydrolysate constituents to re-arrange
411
themselves at the A-W interface. Rullier et al.
412
efficient at forming foams in a non-aggregated state than when present in a heat-induced
413
aggregated form. In contrast, the presence of aggregated proteins led to higher thin film and
414
foam stability. Such effects have also been described for food proteins such as those of pea 33,
415
soy 34, egg white 35 and whey 35, 36. Thus, the presence of aggregated as well as non-aggregated
416
peptides in gluten hydrolysates may contribute to their overall foaming characteristics. From
417
the results in Figure 6, no direct relationship between the aggregation state of the hydrolysates
418
at different pH values and their A-W interfacial behavior (see section 3.3) could be established.
419
However, given the relatively large variation of the particle size distributions (as described
420
above), it is likely that the aggregates observed here are not very compact and that they re-
421
arrange or dissociate in the whipping procedure to produce foams. A more systematic study on
422
the role of controlled aggregation of protein hydrolysates on the way they stabilize A-W
423
interfaces is desirable.
31, 32
showed that β-lactoglobulins are more
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424
In conclusion, depending on the enzyme used for their production and the degree of hydrolysis
425
reached, different gluten hydrolysates were affected in a very different way by changes in pH.
426
At pH 7.0, DH 2 hydrolysates had better FS than DH 6 hydrolysates, while at pH 3.0, tryptic
427
hydrolysates had better FS than peptic hydrolysates. At pH 5.0, which was close to the PZC of
428
the hydrolysates, they all had relatively high E’ values, but very low FS. We believe this is due to
429
a lack of electrostatic repulsion between gas bubbles, making their coalescence more likely,
430
irrespective of the strength of the protein films. At pH 3.0 and 7.0 the hydrolysates had an
431
overall positive or negative charge so there was some repulsive effect between gas bubbles. At
432
these pH values, protein film elasticity values were in line with their FS. Thus, surface charge
433
was a major factor which directly contributed to FS in the proximity of the PZC. At other pH
434
values, FS depended on the strength and integrity of the protein films at the A-W interface, to
435
which hydrophobic interactions probably contributed significantly. While pH-induced changes in
436
protein conformation for native (plant) proteins have been studied in various instances, prior to
437
the present work this was not the case for protein hydrolysates. Surface hydrophobicity and
438
intrinsic fluorescence measurements performed here did in fact suggest that changes in protein
439
conformation took place in gluten hydrolysates when varying the pH. Such changes seemed
440
most pronounced when changing the pH to 3.0, which is the pH value where striking differences
441
in FS between peptic and tryptic hydrolysates were observed. However, at this pH, there was a
442
clear distinction neither in surface hydrophobicity nor in or fluorescence intensity between
443
tryptic and peptic hydrolysates. It is of course possible that different conformational re-
444
arrangements lead to a similar response in fluorescence intensity, but with a completely
445
different effect on interfacial stabilization. At the same time, the techniques used here only
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446
allow evaluating conformational changes of the hydrolysates in solution, while peptide behavior
447
at the A-W interface might still be different. Furthermore, it is important to consider protein
448
hydrolysate aggregation, as it became clear that hydrolyzed gluten formed relatively large but
449
probably loose aggregates which may influence foaming. Such aggregates can to a certain
450
extent be considered ‘particles’. Upon adsorption at an A-W interface, inorganic particles
451
provide excellent stability against disproportionation and coalescence by forming a strong
452
mechanical barrier 37. However, research on biodegradable, food-grade, protein-based particles
453
has only recently emerged and much is still to be learned
454
assess the role of peptide aggregation on the A-W interfacial and foaming behavior of protein
455
hydrolysates.
456
Finally, we conclude that all measurements performed here were necessary to better
457
understand how structural properties of plant protein hydrolysates change with pH, and how
458
this is related to their foaming behavior. Still, not all observations could be explained,
459
illustrating the complexity of studying the behavior of proteins at the A-W interface.
38-40
. More research is needed to
460 461 462 463
ABBREVIATIONS USED
464
FC, foaming cacapity; FS, foam stability; A-W, air-water; ZP, zeta potential; DH, degree of
465
hydrolysis; h, number of hydrolyzed peptide bonds; htot, total number of peptide bonds present;
466
Mp, mass of protein; Mx, molarity of acid or base; meqv, milli-equivalents; SDS, sodium dodecyl
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467
sulphate; ANS, 1-anilino-8-naphthalene sulphonic acid; PSD, particle size distribution; t, time;
468
PZC, point-of-zero-charge.
469 470
ACKNOWLEDGEMENTS
471
I. Rombouts thanks the Research Foundation – Flanders (FWO, Brussels, Belgium) for financial
472
support. K. Brijs acknowledges the Industrial Research Fund (KU Leuven, Leuven, Belgium) for a
473
position as Industrial Research Manager. J.A. Delcour is W.K. Kellogg Chair in Cereal Science and
474
Nutrition at KU Leuven. This work is part of the Methusalem program “Food for the Future” at
475
KU Leuven.
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476
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23. Nielsen, P. M.; Petersen, D.; Dambmann, C. Improved method for determining food protein degree of hydrolysis. J Food Sci. 2001, 66, 642-646.
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24. Caessens, P. W. J. R.; Gruppen, H.; Visser, S.; van Aken, G. A.; Voragen, A. G. J. Plasmin hydrolysis of beta-casein: Foaming and emulsifying properties of the fractionated hydrolysate. J Agric Food Chem. 1997, 45, 2935-2941.
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26. Wouters, A. G. B.; Rombouts, I.; Legein, M.; Fierens, E.; Brijs, K.; Blecker, C.; Delcour, J. A. Air–water interfacial properties of enzymatic wheat gluten hydrolyzates determine their foaming behavior. Food Hydrocolloid. 2016, 55, 155-162.
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30. Ghisaidoobe, A.; Chung, S. Intrinsic Tryptophan Fluorescence in the Detection and Analysis of Proteins: A Focus on Förster Resonance Energy Transfer Techniques. Int J Mol Sci. 2014, 15, 22518–22538.
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31. Rullier, B.; Axelos, M. A. V.; Langevin, D.; Novales, B. beta-Lactoglobulin aggregates in foam films: Correlation between foam films and foaming properties. J Colloid Interface Sci. 2009, 336, 750-755.
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32. Rullier, B.; Novales, B.; Axelos, M. A. V. Effect of protein aggregates on foaming properties of β-lactoglobulin. Colloids Surf Physicochem Eng Aspects. 2008, 330, 96-102.
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33. Liang, H.-N.; Tang, C.-h. Pea protein exhibits a novel Pickering stabilization for oil-inwater emulsions at pH 3.0. LWT - Food Sci Technol. 2014, 58, 463-469.
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34. He, Z. Y.; Li, W. W.; Guo, F. X.; Li, W. Y.; Zeng, M. M.; Chen, J. Foaming Characteristics of Commercial Soy Protein Isolate as Influenced by Heat-Induced Aggregation. Int J Food Prop. 2015, 18, 1817-1828.
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35. Nicorescu, I.; Vial, C.; Talansier, E.; Lechevalier, V.; Loisel, C.; Della Valle, D.; Riaublanc, A.; Djelveh, G.; Legrand, J. Comparative effect of thermal treatment on the physicochemical properties of whey and egg white protein foams. Food Hydrocolloid. 2011, 25, 797-808.
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36. Nicorescu, I.; Riaublanc, A.; Loisel, C.; Vial, C.; Djelveh, G.; Cuvelier, G.; Legrand, J. Impact of protein self-assemblages on foam properties. Food Res Int. 2009, 42, 1434-1445.
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37. Binks, B. P. Particles as surfactants—similarities and differences. Curr Opin Colloid Interface Sci. 2002, 7, 21-41.
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38. Xiao, J.; Li, Y.; Huang, Q. Recent advances on food-grade particles stabilized Pickering emulsions: Fabrication, characterization and research trends. Trends Food Sci Technol. 2016, 55, 48-60.
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39. Lam, S.; Velikov, K. P.; Velev, O. D. Pickering stabilization of foams and emulsions with particles of biological origin. Curr Opin Colloid Interface Sci. 2014, 19, 490-500.
611 612 613
40. Tavernier, I.; Wijaya, W.; Van der Meeren, P.; Dewettinck, K.; Patel, A. R. Food-grade particles for emulsion stabilization. Trends Food Sci Technol. 2016, 50, 159-174.
614 615 616
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617
FIGURE CAPTIONS
618
Figure 1: Impact of pH on the foaming properties of 0.050% (wprotein/v) solutions of T2, T6, P2
619
and P6 hydrolysates. The codes T and P refer to gluten trypsin and pepsin hydrolysates, the
620
codes 2 and 6 refer to degrees of hydrolysis of 2 and 6.
621 622
Figure 2: Impact of pH on the decrease of surface tension at the air-water (A-W) interface after
623
forming a hanging drop with 0.050% (wprotein/v) solutions of T2, T6, P2 and P6 hydrolysates.
624
After 10 min, the drops were sinusoidally oscillated to determine surface dilatational moduli
625
(E’). Codes T2, T6, P2 and P6 as in Figure 1.
626 627
Figure 3: Impact of pH on the zeta potential (ZP) of 0.15% (wprotein/v) solutions of T2, T6, P2 and
628
P6 hydrolysates. Codes T2, T6, P2 and P6 as in Figure 1.
629 630
Figure 4: Impact of pH on the surface hydrophobicity of solutions of T2, T6, P2 and P6
631
hydrolysates. Codes T2, T6, P2 and P6 as in Figure 1.
632 633
Figure 5: Impact of pH on the intrinsic tryptophan fluorescence emission spectra of solutions of
634
T2, T6, P2 and P6 hydrolysates after excitation at 295 nm. Codes T2, T6, P2 and P6 as in Figure 1.
635 636
Figure 6: Impact of pH on the particle size distribution (PSD), measured with dynamic light
637
scattering) of 0.15% (wprotein/v) solutions of T2, T6, P2 and P6 hydrolysates. Codes T2, T6, P2 and
638
P6
as
in
Figure
1. 28
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TABLE 1
Table 1: Impact of pH on the protein solubility of T2, T6, P2 and P6 hydrolysates. Capital letters indicate significant (p < 0.05) differences between different pH values for a given sample. Lowercase letters indicate significant (p < 0.05) differences between samples at a given pH value. The codes T and P refer to gluten trypsin and pepsin hydrolysates, the codes 2 and 6 refer to degrees of hydrolysis of 2 and 6.
pH 3.0
pH 5.0
pH 7.0
T2
99 ± 3 A,ab
95 ± 2 A,a
99 ± 0 A,a
T6
97 ± 1 A,ab
94 ± 2 A,a
98 ± 1 A,a
P2
101 ± 1 A,a
97 ± 1 B,a
98 ± 0 B,a
P6
95 ± 1 A,b
96 ± 1 A,a
94 ± 1 A,b
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TABLE 2 Table 2: Estimation of equilibrium surface tension of 0.050% (wprotein/v) solutions of T2, T6, P2 and P6, at pH 3.0, 5.0 and 7.0, at t ∞ based on surface tension vs. 1/√t plots using the same data as displayed in Figure 2. Capital letters indicate significant (p < 0.05) differences for a given sample at different pH values. Lowercase letters indicate significant (p < 0.05) differences between samples at a given pH value. Codes T2, T6, P2 and P6 as in Table 1.
pH 3.0
pH 5
pH 7
T2
48.6 ± 0.5 B,d
49.0 ± 1.0 B,c
51.1 ± 0.6 A,ab
T6
50.8 ± 0.4 A,c
50.0 ± 0.7 B,b
50.4 ± 0.5 AB,b
P2
54.4 ± 1.3 A,b
49.7 ± 0.3 B,bc
49.5 ± 0.5 B,c
P6
58.7 ± 0.7 A,a
52.1 ± 0.6 B,a
51.8 ± 0.9 B,a
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FIGURE 1
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FIGURE 2
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FIGURE 3
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FIGURE 4
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FIGURE 5
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FIGURE 6
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² TABLE OF CONTENT GRAPHIC
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