Food Hydrocolloids 75 (2018) 223e228

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Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd

Effect of oxidative modification on structural and foaming properties

of egg white protein
Xiang Duan a, Mei Li a, Jing Shao a, Haiying Chen b, Xueming Xu b, Zhengyu Jin b,
Xuebo Liu a, *
a
College of Food Science and Engineering, Northwest A&F University, Yangling 712100, PR China
b
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, PR China

a r t i c l e i n f o a b s t r a c t

Article history: Protein oxidation results in structural modification which affects its functionalities. In this study, 2,20 -
Received 20 April 2017 azobis (2-amidinopropane) dihydrochloride (AAPH) was selected as a representative of lipid peroxida-
Received in revised form tion products to investigate the effect of oxidative modification on structural and foaming properties of
24 July 2017
egg white protein. Incubation of egg white protein with AAPH resulted in structural changes, regarding
Accepted 9 August 2017
Available online 13 August 2017
by sulfhydryl-disulfide interchange reaction and increase in dityrosine formation. Moderate oxidation led
to exposure of hydrophobic groups of egg white protein, whereas, excessive oxidation induced a decrease
in surface hydrophobicity. Molecular weight distribution and scanning electron microscopy showed that
Keywords:
Egg white protein
egg white protein underwent an aggregation after excessive oxidation. Furthermore, after treating with
Oxidation AAPH at 0.2 mM, the foaming ability of egg white protein increased from 85.6% to 91.4%, and the foaming
Structural characteristics stability increased from 80.6% to 85.3%. The excessive oxidation treatment (5 and 25 mmol/L AAPH)
Foamability resulted in an enhanced foaming ability, while reduced its foaming stability. This study suggested that
oxidation treatment has a potential to be implemented to modify foaming properties of egg white
protein.
© 2017 Published by Elsevier Ltd.

1. Introduction
Because of its high foaming property, egg white protein is
widely used as a foaming agent in food industry to improve and
maintain the quality (texture and volume) of aerated food, such as
cakes, cookies, dessert shells and chocolate mousses (Huang, Tu
et al., 2017). Foams and bubbles play a very important role in
these food products in terms of their structure and texture. During
these food processing, egg white protein can encapsulate and retain
air, and thus increase foam volume. Therefore, enhancing foaming
ability of egg white protein is significant for improving quality of
egg protein-based food products.
The adsorption of protein at air-water interface is known to play
a key role in formation and stability of food dispersed system as
foams (Morales, Martínez, Ruiz-Henestrosa, & Pilosof, 2015). When
air comes into egg solution, egg protein can adsorb at air-water
interface and undergo rapid conformational change to form
* Corresponding author.
E-mail address: xueboliu@nwsuaf.edu.cn (X. Liu).
cohesive viscoelastic films (Campbell, Raikos, & Euston, 2003). The
conformational state of protein strongly affected adsorption and
reorganization of protein layer at air-water interface (Jarpa-Parra
et al., 2015). For some proteins, unfolded molecules may adsorb
more easily to air bubble surface, and thus exhibited enhanced
foaming functionality (Mirmoghtadaie, Aliabadi, & Hosseini, 2016).
Previous researches have demonstrated that denatured ovalbumin
possessed a higher foaming power than native ovalbumin due to
the increased flexibility and surface hydrophobicity (Graham &
Phillips, 1976). Thus, a certain degree of structural change might
have a beneficial impact on egg white protein foam formation and
stabilization.
During food processing, proteins are often exposed to oxidizing
conditions and are vulnerable to oxidative modification (Wu, Hou,
Zhang, Kong, & Hua, 2009). Oxidative stress results in multiple
structural changes of proteins, including oxidation of side chain
groups, backbone fragmentation, unfolding, degradation and ag-
gregation (Huang, Sontag-Strohm, Stoddard, & Kato, 2017). Recent
studies reported that oxidation treatment could improve functional
characteristics (heat stability and gel properties) of whey and egg
white proteins via changing their structures (Sutariya & Patel, 2017;

http://dx.doi.org/10.1016/j.foodhyd.2017.08.008
0268-005X/© 2017 Published by Elsevier Ltd.
224 X. Duan et al. / Food Hydrocolloids 75 (2018) 223e228
Takahashi, Handa, Yamaguchi, Kodama, & Chiba, 2016). In this
work, we are interested in whether oxidation treatment could be
used to improve foaming ability of egg white protein.
In food system, protein oxidation and lipid peroxidation are
usually interdependent of each other. During food processing, lipid-
derived peroxyl radicals can covalently modified side chains and
polypeptide backbone of protein molecules, resulting in confor-
mational changes of proteins (Wu, Hua, & Lin, 2014). The water-
soluble radical initiator 2,20 -azobis (2-amidinopropane) dihydro-
chloride (AAPH) could initiate peroxyl radicals, which are respon-
sible for oxidation of proteins. In previous researches, oxidative
modification of proteins by AAPH-derived peroxyl radicals has been
extensively studied (Fuentes-Lemus et al., 2016). However, little
information is available on the oxidation of egg white protein by
peroxyl radicals. In this paper, egg white protein was incubated
with increasing concentration of AAPH, and the changes in struc-
tural and foaming properties of egg white protein were
determined.
The relationship between changes in structural properties and
foaming properties of egg white protein during oxidation treat-
ment was elucidated by determining free sulfhydryl groups (SH),
surface hydrophobicity (Ho), secondary structures, molecular
weight (MW) distribution, morphology and foaming properties as a
function of oxidation degree.
2. Materials and methods
2.1. Materials
Hen eggs were purchased from a local market. AAPH, 1-anilino-
8-naphthalene-sulphonate (ANS), 5,50 -dithiobis (2-nitrobenzoate)
(DTNB), the protein standards for studying MW distribution,
including bovine serum albumin (66,000 Da), carbonic anhydrase
(29,000 Da) and aprotinin (6500 Da) were purchased from Sigma-
Aldrich Co. LLC. (Shanghai, China). All other chemicals were of
analytical reagent grade.
2.2. Egg white protein oxidation
Egg white was separated from yolk and stirred gently using a
magnetic stirrer (IKA, IKA Works Inc., Wilmington, NC, USA) so as
not to produce foam for 1 h at 4  C, giving homogeneous egg white.
After that, the egg white was lyophilized and stored at 20  C until
use (Kakalis & Regenstein, 1986). The oxidative modification of egg
white protein was processed according to the method described by
Wu et al. (2009). Briefly, the egg white dispersion (10 mg/mL,
suspended in 0.01 mol/L sodium phosphate buffer, pH 7.4) was
mixed with a serial concentrations of AAPH, and then incubated by
continuous shaking under air at 37  C in dark for 24 h. The final
concentration of AAPH was zero (control), 0.04, 0.2, 1, 5, and
25 mM. The reaction was stopped by immediately cooling the
dispersions to 0e4  C by ice-bathing. After centrifuging at 5000 g
for 1 h at 4  C to remove small quantity of insoluble substances
which were formed during solution cooling, the protein dispersions
were dialyzed against deionized water at 4  C for 72 h to remove
residual AAPH and then were freeze-dried and stored at 20  C.
2.3. Determination of dityrosine formation
Dityrosine formation was estimated by the method of Morzel,
Gatellier, Sayd, Renerre, and Laville (2006). Weighed amounts of
freeze-dried samples (10 mg) were dissolved in 5 mL of high ionic
buffer (20 mM phosphate buffer at pH 6 containing 0.6 M KCl). The
protein concentration was estimated by the Biuret method (Kakalis
& Regenstein, 1986). Dityrosine content was estimated by
fluorescent measurement at 420 nm after excitation at 325 nm,
using a Hitachi F4500 fluorescence spectrometer (Hitachi Ltd.,
Tokyo, Japan). Corrected fluorescence was obtained by dividing
measured fluorescence by protein concentration. The results were
expressed in arbitrary units.
2.4. Free SH group content
Free SH group content was measured according to the method
of Li, Zhu, Zhou, and Peng (2012). Ellman’s reagent was prepared by
dissolving 40 mg of DTNB in 10 mL of Tris-Gly buffer (10.4 g of Tris,
6.9 g of Gly, and 1.2 g of disodium ethylenediaminetetraacetic acid
(EDTA) in 1000 mL of deionized water, pH 8.0). The lyophilized
proteins were dissolved with Tris-Gly-SDS buffer (45 mL Tris-Gly
buffer containing 5 mL of 2.5% (wt/v) SDS aqueous solution) to a
final protein concentration of 10 mg/mL. Four milliliter of each
sample was mixed with 0.04 mL of Ellman’s reagent solution.
Subsequently, the mixtures were shaken and incubated in dark at
room temperature for 10 min. The absorbance of supernatant was
measured at 412 nm against the blank (without Ellman’s reagent
and sample). Absorbance values were converted to amounts of free
SH groups using a calibration curve with reduced glutathione
(Wang, Chen, et al., 2014).
2.5. Surface hydrophobicity (Ho)
Ho of proteins was determined by following the method of
Wang, Chen, et al. (2014), Wang, Tao, et al. (2014), with slight
modifications. Each sample was diluted to five concentrations be-
tween 0.0% and 0.1% protein (wt/wt) in 10 mM phosphate buffer
(pH 7.0). Then, aliquots (20 mL) of ANS (8.0 mM in the same buffer)
were added to 4 mL of protein solutions. The solutions were rested
for 3 min in dark and placed in the cell of a Hitachi F4500 fluo-
rescence spectrometer (Tokyo, Japan), and the fluorescence in-
tensity was measured at 470 nm, using excitation at 390 nm, slit
width 5 nm. Appropriate blanks corresponding to the buffer were
subtracted to correct background fluorescence. Ho was determined
according to the slope method of Alizadeh-Pasdar and Li-Chan
(2000).
2.6. Secondary structures
The change in secondary structures of egg white protein was
determined using Fourier transform infrared spectrophotometer
(FTIR, Bruker, Germany). The protein samples were mixed with KBr
in ratio of 1:200 and ground in a mortar by hand with a pestle. The
powders were pressed into thin sheets under a pressure of 4 t. The
infrared radiation absorbency scans were analyzed in range of
4000-400 cm1 for changes in intensity of sample peaks, with air as
background. The spectra were analyzed with Omnic software
package (version 6.1a, Thermo Nicolet Corp) and PeakFit v4.12
(SYSTAT Software inc.).
2.7. MW distribution
The MW distribution profiles of egg white protein were esti-
mated according to the method described by Liu et al. (2012). A
Waters 600 liquid chromatography system (Waters Co., Milford,
MA, USA) equipped with a 2487 UV detector was used for this
experiment. The UV detector was set at 215 nm. The column used
was a silica-based column (TSK G3000SWXL, 300  6.5 mm, Tosoh
Co., Tokyo, Japan), whereas the mobile phase consisting of PBS was
delivered at a flow rate of 0.5 mL/min, and 20 mL of samples were
injected into the high-performance liquid chromatography (HPLC)
system. A MW calibration curve was obtained from following
 X. Duan et al. / Food Hydrocolloids 75 (2018) 223e228
standards: bovine serum albumin (66,000 Da), carbonic anhydrase
(29,000 Da), and aprotinin (6500 Da).
2.8. Scanning electron microscope (SEM)
The morphology of egg white protein was observed with a SEM.
The lyophilized samples were sputter-coated with gold, and
examined in a Hitachi S-3400N scanning electron microscope
(Hitachi, Tokyo, Japan) at 5.0 kV.
2.9. Foaming properties
Foam formation and collapse were analyzed using the Dynamic
Foam Analyzer DFA100 (Krüss GmbH, DE) as given in Dombrowski,
Johler, Warncke, and Kulozik (2016). Foaming properties are ob-
tained by observing the change in foam volume as well as liquid
volume over time by an optical sensor. Foams were prepared by air
sparging (t ¼ 6 s, Ṽ ¼ 0.3 L/min) through a porous glass frit into the
provided protein solutions (V ¼ 50 mL). Immediately following,
foam decay performance was recorded for a time period of 10 min.
The obtained data on time-dependent evolution of foam as well as
liquid height was used to calculate foamability (FA), the proportion
of initial liquid volume integrated into the foam volume (RL) and
foam stability (FS) as follows:
Vfoam; max
FA ¼  100%
Vliquid; i
Vliquid; foam max
RL ¼  100%
Vliquid; i
Vfoam; t¼x
FS ¼  100%
Vfoam; max
Here, Vfoam, max is the maximum foam volume after air sparging into
the protein solution with Vliquid,i ¼ 50 mL. Vliquid, foam max is the liquid
volume bound in the foam at the time of maximum foam volume.
Vfoam,t¼x is the remaining foam volume after a respective decay time
x (x ¼ 10 min).
In addition, foam liquid stability (FLS) is the time dependent
percentage of the actual liquid volume bound in the foam with
respect to the maximum liquid volume bound in the foam: Vliquid,
t¼x/Vliquid, foam max. And the tFLS 75 is the time at which FLS has
reduced to 75% of its initial value.
2.10. Statistical analysis
The experiments were run in triplicate. The results were
expressed as the mean ± standard deviation and were analyzed
using SPSS 16.0 software (SPSS, Inc., Chicago, IL, USA). One-way
analysis of variance (ANOVA) was used to analyze data, and Dun-
can’s multiple-range tests were conducted to determine differences
between the means. Results were considered statistically signifi-
cant at P < 0.05.
3. Results and discussion
3.1. Dityrosine formation
During protein oxidation, exogenous tyrosine phenoxyl radicals
can form dityrosines on proteins. Dityrosine production has been
described as an useful oxidation parameter for protein modification
(Zhang, Xiao, & Ahn, 2013). In our experiment, a significant increase
in dityrosine formation was observed when egg white protein was
225
exposed to 1.0 mM and higher AAPH (Table 1). After exposure to
AAPH at 25 mM, the level of dityrosine was 21.48, representing
almost 3-fold increase over that found in the untreated sample.
Reactions of peroxyl radicals with protein molecule result in
structural modification of side chain groups. Side chain groups of
tyrosine, cysteine, tryptophan, methionine, proline, valine, histi-
dine, and lysine residues were vulnerable to peroxyl radicals (Wu,
Zhang, et al., 2009). The result showed that the dityrosine con-
tent increased with increasing AAPH concentration, suggesting that
the oxidation degree of egg white protein increased gradually with
increasing AAPH concentration.
3.2. Free SH group content
The interfacial activity and ability to form protein adsorption
layers are partly governed by conformation of protein at water-air
interface (Fischer, 2013). Molecular alterations induced by reduc-
tion of disulphide linkages among protein molecules improved film
formation in foam (Alleoni, 2006). In this work, the free SH contents
in egg white with different oxidation degrees are shown in Table 1.
The free SH content of untreated sample was 41.06 mmol/g protein.
After oxidation treated with low concentration of AAPH (0.04 and
0.2 mM), the free SH contents increased to 47.87 and 65.46 mmol/g
protein, respectively. This was attributed to cleavage of protein
disulfides, and this meant that the breakup of disulfide bonds was
more prominent than the formation of new ones by thiol oxidation
at low AAPH concentration. Whereas, when concentration of AAPH
increased to 1.0 mM or higher, the free SH contents decreased to the
values which were similar to that of untreated sample. This was in
agreement with previous study reporting that high peroxyl radicals
could react with sulphydryl groups to form sulphinyl radicals and
decrease free SH group content (Wu, Zhang, et al., 2009). Among
egg white proteins, ovalbumin is the only protein that has free -SH
groups, containing a single disulfide bond between Cys74 and
Cys121. Ovotransferrin and lysozyme contain 15 and 4 disulfide
bonds, separately. Ovomucoid molecule is composed of three
distinct domains crosslinked only by intradomain disulfide bonds.
Ovomucin was considered as a biopolymer which was cross-linked
by disulfide bonds (Mine, 2014). These free SH and disulfide bonds
contribute to stability of egg white protein structures. The above
result indicated that oxidation treatment caused sulfhydryl-
disulfide interchange reaction between disulfide groups, which
would induce alterations of structural and functional properties of
egg white protein.
3.3. Surface hydrophobicity (Ho)
The affinity of surface active molecules to be adsorbed at air-
water interface is from their special amphiphilic structure
(Karbaschi et al., 2014). Ho is a common characteristic to evaluate
structural change of proteins (Wang, Tao, et al., 2014). Table 1
shows Ho values of egg white with different oxidation degrees. It
could be seen that incubation of egg white protein with AAPH
resulted in an increase in Ho values, except for AAPH at 25 mM. This
suggested that moderate oxidation led to protein partial unfolding
and caused hydrophobic groups buried inside molecules to be
exposed. The spontaneous adsorption of proteins from solution to
air interface is important to their foaming properties. Adsorption is
favored by exposition of hydrophobic residues, and protein mole-
cules with higher Ho values exhibited a higher adsorption rate
(Jarpa-Parra et al., 2015).
3.4. Secondary structure
FTIR analysis was applied to investigate the change in secondary
226 X. Duan et al. / Food Hydrocolloids 75 (2018) 223e228

Table 1
Dityrosine content, free sulfhydryl (SH) group content and surface hydrophobicity (Ho) of egg white protein incubated with increasing concentration of AAPH for 24 h at 37  C.
Different letters (a-d) indicated significant differences (P < 0.05).

AAPH (mM)

0 0.04 0.2 1 5 25

Dityrosine 7.41 ± 0.78d 7.64 ± 0.02d 7.82 ± 0.24d 10.18 ± 0.09c 19.72 ± 0.23b 21.48 ± 0.28a
Free SH (mmol/g protein) 41.06 ± 0.28c 47.87 ± 2.15b 65.46 ± 2.99a 42.50 ± 0.15c 43.02 ± 1.48c 43.89 ± 0.86bc
Ho 410.3 ± 7.1c 565.1 ± 29.7a 498.5 ± 24.5b 467.5 ± 20.7bc 495.4 ± 6.7b 341.6 ± 41.5d

structure of egg white protein during oxidation treatment. Char- Table 2

acteristic bands in FTIR spectra of proteins mainly include amide I Percentage of secondary structures of egg white protein incubated with increasing
concentration of AAPH from Fig. 2. Different letters (a-b) in the same column indi-
(1600-1700 cm1). Studies with proteins of known structure have cated significant differences (P < 0.05).
been used to correlate systematically the shape of amide I band to
secondary structure content (Byler & Susi, 1986). It could be seen AAPH (mM) Secondary structures (%)

that the secondary structure of egg white protein was composed of a-helixes b-sheets b-turns
three different conformations including a-helixes, b-turns and b- 0 28.60 ± 0.01b 38.45 ± 0.07a 32.95 ± 0.07ab
sheets (Fig. 1). The percentages of these secondary structures are 0.04 44.29 ± 0.89a 28.60 ± 0.12a 32.87 ± 0.76ab
summarized in Table 2. The b-sheets were dominant in native 0.2 36.08 ± 0.02ab 35.06 ± 0.13a 28.87 ± 0.13b
± ± ±
sample (38.45%), and no significant difference was observed in b- 1 38.79 11.20ab 24.94 11.89a 36.28 0.69a
5 41.72 ± 3.83ab 25.02 ± 6.68a 33.28 ± 2.86a
sheets proportion after oxidative modification (P > 0.05). Mean- 25 40.31 ± 3.90ab 27.02 ± 6.72a 32.68 ± 2.81ab
while, the proportion of a-helixes significantly increased after in-
cubation with AAPH at 0.04 mM. The presence of a-helixs structure
could contribute to formation of more stable interfaces and
multilayer interfaces, likely due to possible creation of more non- 3.5. MW distribution
covalent intramolecular interactions (Jarpa-Parra et al., 2015).
This result showed that the secondary structures of egg white The size exclusion chromatogram using a HPLC system was used
protein were sensitive to peroxyl radicals, and egg white protein to study MW distribution profiles of oxidized egg white protein,
exhibited different structural behaviors in different oxidative and the results are shown in Fig. 2 and Table 3. Egg white presented
environments. a polydisperse distribution with two major peaks of 60 kDa and
21 kDa. After oxidation treatment, little change was observed in
MW distribution when the concentration of AAPH increased from
0.04 to 5 mM. When the concentration of AAPH increased to
25 mM, the percentage of high MW fraction largely increased to
83.64%. Correspondingly, the percentage of low MW fraction
decreased to 16.36%. This phenomenon implied that treatment
with high concentration of AAPH (25 mM) induced egg white
protein aggregation, which was in agreement with previous study
reporting that excessive oxidation resulted in denaturation and
aggregation of soy proteins (Liang, 1999). Aggregation is a common

Fig. 2. High-performance size exclusion chromatograph of egg white protein incu-

Fig. 1. FTIR spectra of egg white protein incubated with different concentration of bated with different concentration of AAPH for 24 h at 37  C. AeF: the chromatograms
AAPH for 24 h at 37  C. aef: the deconvolution in amide I band of egg white protein of egg white protein incubated with 0, 0.04, 0.2, 1, 5 and 25 mM AAPH, respectively.
incubated with 0, 0.04, 0.2, 1, 5 and 25 mM AAPH, respectively. The column was a silica-based column (TSK G3000SWXL, 300  6.5 mm).
 X. Duan et al. / Food Hydrocolloids 75 (2018) 223e228 227

Table 3 Table 4
Percentage area of peaks of high-performance size exclusion chromatograph of egg Foamability (FA), proportion of initial liquid volume integrated into the foam
white protein incubated with increasing concentration of AAPH for 24 h at 37  C. structure (RL), foam stability (FS) and the time at which FLS has reduced to 75% (tFLS
Different letters (a-b) in the same column indicated significant differences (P < 0.05). 75%) of egg white proteins in dependence of concentration of AAPH. Different letters
(a-e) in the same column indicated significant differences (P < 0.05).
AAPH (mM) Percentage area of peak (%) corresponding retention time
AAPH (mM) FA (%) RL (%) FS (%) tFLS 75% (s)
12-15 min (about 60 kDa) 15-20 min (about 21 kDa)
0 85.6 ± 2.2d 25.0 ± 1.7bc 80.6 ± 0.6bc 19.2 ± 2.3a
0 78.17 ± 3.85b 21.83 ± 1.67a
0.04 89.8 ± 3.1cd 22.6 ± 3.4c 83.5 ± 1.1ab 20.1 ± 3.6a
0.04 77.93 ± 3.44b 22.07 ± 1.52a
0.2 91.4 ± 2.5c 23.4 ± 1.8c 85.3 ± 2.3a 17.9 ± 4.0a
0.2 78.21 ± 3.19b 21.79 ± 1.73a
1 90.4 ± 2.1cd 25.6 ± 2.4bc 77.0 ± 2.0cd 22.1 ± 3.1a
1 78.86 ± 2.80b 21.14 ± 1.61a
5 98.0 ± 2.7b 29.8 ± 3.5b 75.1 ± 2.9d 18.8 ± 2.6a
5 77.93 ± 3.05b 22.07 ± 1.14a
25 117.0 ± 3.a 48.6 ± 2.7a 50.1 ± 2.4e 9.1 ± 1.6b
25 83.64 ± 3.22a 16.36 ± 1.38b

at 0.2 mM, the FA increased from 85.6% to 91.4% compared with

phenomenon for peroxyl radical-mediated protein oxidation (Wu,
untreated sample. The FA of proteins is fundamentally related to
Zhang, et al., 2009). The mechanism by which aggregates
their properties of forming films in air-water interface. The in-
contribute to foam properties is not completely elucidated. Some
crease in FA after moderate oxidation (0.2 mM AAPH) was
studies showed that non-aggregated proteins could adsorb to air-
attributed to unfolded structure and exposure of hydrophobic
water interface more rapidly than protein aggregates (Rullier,
groups, which enhanced protein-air interactions. In addition,
Novales, & Axelos, 2008). Whereas, some studies reported that
excessive oxidation (5 and 25 mM AAPH) also induced a signif-
certain aggregates possessed enhanced ability to form cohesive
icant increase in FA, increasing to 98.0% and 117.0%, respectively.
viscoelastic interfacial films (Amagliani & Schmitt, 2017).
Meanwhile, the RL value increased largely from 25.0% to 29.8%
and 48.6% after treatment with 5 and 25 mM AAPH, respectively.
3.6. Morphology
This meant that excessive oxidation induced more liquid volume
bound in the foam compared with untreated sample. Combined
To have tangible evidence of transformation of egg white pro-
with above-mentioned results, this might be due to aggregation
tein microstructure, we employed SEM (Fig. 3). It could be seen that
of protein, which enhanced its ability to adsorb at air-water
the untreated sample exhibited a morphology characteristic
interface.
without fibrillar or sheet-like structures (Fig. 3a). After oxidative
The FS of egg white protein increased from 80.6% to 85.3% after
modification, the arrangement changed with increasing oxidation
oxidation treatment with 0.2 mmol/L AAPH (Table 4). This result
degree. At the stage of moderate oxidation (AAPH below 1 mmol/L),
was in agreement with recent research reporting that unfolding of
the sheet-like structures were observed after oxidation treatment
soy protein isolate peptide chains could make the interaction be-
(Fig. 3bed). This alteration was because that oxidation treatment
tween hydrophobic residues stronger than natural state and thus
led to unfolding of protein molecules and increased exposure of
increase foaming stability (Wang et al., 2016). Contrarily, the FS
hydrophobic groups (Wang, Li, Yan, Huang, & Dong, 2016), which
reduced when the concentration of AAPH increased to 5 and
enhanced interaction between proteins and then gave them more
25 mmol/L, decreasing to 75.1% and 50.1%, respectively (Table 4).
ordered arrangement. Furthermore, excessively oxidized egg white
For a better comparability of the obtained results, the tFLS 75%, which
protein (AAPH at 5 and 25 mmol/L) formed network composing of
refers to the time period of reaching 25% liquid drainage, was
randomly aggregated particles and granules with a thick and dense
determined. It could be seen that the tFLS 75% dramatically decreased
structure (Fig. 3 eef), supporting the MW distribution result
from 19.2 s to 9.1 s after oxidation treatment with 25 mmol/L AAPH
showing that the percentage of high MW fraction increased after
(Table 4). This meant that unstable bubbles in the rising foam easily
excessive oxidation.
coalesced or collapsed because the interfacial adsorption of
excessively oxidized protein was weakened. Combined with
3.7. Foaming properties aforementioned results, the main reason for this behavior was the
decreased in Ho and larger aggregates, which caused steric im-
Foaming behavior of egg white protein with different oxida- pediments and thus did not allow for the formation of a closely
tion degrees is shown in Table 4. For sample treated with AAPH packed interfacial layer (Amagliani & Schmitt, 2017).

4. Conclusion

In this work, the structural and foaming properties of egg

Fig. 3. The morphology of egg white protein incubated with different concentration of
AAPH for 24 h at 37  C. aef: the SEM images (1.0 k  magnification) of egg white
protein incubated with 0, 0.04, 0.2, 1, 5 and 25 mM AAPH, respectively. The lyophilized
samples were examined in a Hitachi S-3400N at 5.0 kV.
white protein subjected to oxidation treatment were investigated.
The results indicated that oxidation treatment could modify mo-
lecular arrangement and interaction of egg white protein via
changing their dityrosine formation, sulfhydryl-disulfide inter-
change, surface hydrophobicity and secondary structures. In par-
allel, moderate oxidation treatment (treated with 0.2 mmol/L
AAPH) could enhance foaming properties of egg white protein.
Meanwhile, excessive oxidation treatment (25 mmol/L AAPH)
resulted in a larger aggregation, protein oxidation at this stage
would increase the foaming ability of egg white protein, while
decrease its foam stability. These results suggested that oxidation
treatment might be a useful approach to modify foaming ability of
egg white protein.
228 X. Duan et al. / Food Hydrocolloids 75 (2018) 223e228
Acknowledgments
This work was supported by grants from the National Key
Research and Development Program of China (No.
2016YFD0400601) and the National Natural Science Foundation of
China (Nos. 31501426 and 31401493).
References
Alizadeh-Pasdar, N., & Li-Chan, E. C. (2000). Comparison of protein surface hydro-
phobicity measured at various pH values using three different fluorescent
probes. Journal of Agricultural and Food Chemistry, 48(2), 328e334.
Alleoni, A. C. C. (2006). Albumen protein and functional properties of gelation and
foaming. Scientia Agricola, 63(3), 291e298.
Amagliani, L., & Schmitt, C. (2017). Globular plant protein aggregates for stabiliza-
tion of food foams and emulsions. Trends in Food Science & Technology, 67,
248e259.
Byler, D. M., & Susi, H. (1986). Examination of the secondary structure of proteins by
deconvolved FTIR spectra. Biopolymers, 25(3), 469e487.
Campbell, L., Raikos, V., & Euston, S. R. (2003). Modification of functional properties
of egg-white proteins. Food/Nahrung, 47(6), 369e376.
Dombrowski, J., Johler, F., Warncke, M., & Kulozik, U. (2016). Correlation between
bulk characteristics of aggregated b-lactoglobulin and its surface and foaming
properties. Food Hydrocolloids, 61, 318e328.
Fischer, P. (2013). Rheology of interfacial protein-polysaccharide composites. The
European Physical Journal Special Topics, 222(1), 73e81.
Fuentes-Lemus, E., Dorta, E., Escobar, E., Aspee, A., Pino, E., Abasq, M., et al. (2016).
Oxidation of free, peptide and protein tryptophan residues mediated by AAPH-
derived free radicals: Role of alkoxyl and peroxyl radicals. Rsc Advances, 6(63),
57948e57955.
Graham, D., & Phillips, M. (1976). The conformation of proteins at interfaces and their
role in stabilizing emulsions. New York: Academic Press.
Huang, X., Sontag-Strohm, T., Stoddard, F. L., & Kato, Y. (2017). Oxidation of proline
decreases immunoreactivity and alters structure of barley prolamin. Food
Chemistry, 214, 597e605.
Huang, T., Tu, Z.-c., Wang, H., Shangguan, X., Zhang, L., Niu, P., et al. (2017). Pro-
motion of foam properties of egg white protein by subcritical water pre-
treatment and fish scales gelatin. Colloids and Surfaces A: Physicochemical and
Engineering Aspects, 512, 171e177.
Jarpa-Parra, M., Bamdad, F., Tian, Z., Zeng, H., Temelli, F., & Chen, L. (2015). Impact of
pH on molecular structure and surface properties of lentil legumin-like protein
and its application as foam stabilizer. Colloids and Surfaces B: Biointerfaces, 132,
45e53.
Kakalis, L. T., & Regenstein, J. M. (1986). Effect of pH and salts on the solubility of egg
white protein. Journal of Food Science, 51(6), 1445e1447.
Karbaschi, M., Lotfi, M., Kr€ agel, J., Javadi, A., Bastani, D., & Miller, R. (2014). Rheology
of interfacial layers. Current Opinion in Colloid & Interface Science, 19(6),
514e519.
Liang, J.-H. (1999). Fluorescence due to interactions of oxidizing soybean oil and soy
proteins. Food Chemistry, 66(1), 103e108.
Liu, P., Huang, M., Song, S., Hayat, K., Zhang, X., Xia, S., et al. (2012). Sensory char-
acteristics and antioxidant activities of Maillard reaction products from soy
protein hydrolysates with different molecular weight distribution. Food and
Bioprocess Technology, 5(5), 1775e1789.
Li, H., Zhu, K., Zhou, H., & Peng, W. (2012). Effects of high hydrostatic pressure
treatment on allergenicity and structural properties of soybean protein isolate
for infant formula. Food Chemistry, 132(2), 808e814.
Mine, Y. (2014). Egg proteins. Applied Food Protein Chemistry, 459e490.
Mirmoghtadaie, L., Aliabadi, S. S., & Hosseini, S. M. (2016). Recent approaches in
physical modification of protein functionality. Food Chemistry, 199, 619e627.
Morales, R., Martínez, K. D., Ruiz-Henestrosa, V. M. P., & Pilosof, A. M. (2015).
Modification of foaming properties of soy protein isolate by high ultrasound
intensity: Particle size effect. Ultrasonics Sonochemistry, 26, 48e55.
Morzel, M., Gatellier, P., Sayd, T., Renerre, M., & Laville, E. (2006). Chemical oxidation
decreases proteolytic susceptibility of skeletal muscle myofibrillar proteins.
Meat Science, 73(3), 536e543.
Rullier, B., Novales, B., & Axelos, M. A. (2008). Effect of protein aggregates on
foaming properties of b-lactoglobulin. Colloids and Surfaces A: Physicochemical
and Engineering Aspects, 330(2), 96e102.
Sutariya, S., & Patel, H. (2017). Effect of hydrogen peroxide on improving the heat
stability of whey protein isolate solutions. Food Chemistry, 223, 114e120.
Takahashi, M., Handa, A., Yamaguchi, Y., Kodama, R., & Chiba, K. (2016). Anodic
oxidative modification of egg white for heat treatment. Journal of Agricultural
and Food Chemistry, 64(34), 6503e6507.
Wang, P., Chen, H., Mohanad, B., Xu, L., Ning, Y., Xu, J., et al. (2014). Effect of frozen
storage on physico-chemistry of wheat gluten proteins: Studies on gluten-,
glutenin-and gliadin-rich fractions. Food Hydrocolloids, 39, 187e194.
Wang, W., Li, J., Yan, L., Huang, G., & Dong, Z. (2016). Effect of oxidization and
chitosan on the surface activity of soy protein isolate. Carbohydrate Polymers,
151, 700e706.
Wang, P., Tao, H., Wu, F., Yang, N., Chen, F., Jin, Z., et al. (2014). Effect of frozen
storage on the foaming properties of wheat gliadin. Food Chemistry, 164, 44e49.
Wu, W., Hou, L., Zhang, C., Kong, X., & Hua, Y. (2009). Structural modification of soy
protein by 13-hydroperoxyoctadecadienoic acid. European Food Research and
Technology, 229(5), 771e778.
Wu, W., Hua, Y., & Lin, Q. (2014). Effects of oxidative modification on thermal ag-
gregation and gel properties of soy protein by malondialdehyde. Journal of Food
Science and Technology, 51(3), 485e493.
Wu, W., Zhang, C., Kong, X., & Hua, Y. (2009). Oxidative modification of soy protein
by peroxyl radicals. Food Chemistry, 116(1), 295e301.
Zhang, W., Xiao, S., & Ahn, D. U. (2013). Protein oxidation: Basic principles and
implications for meat quality. Critical Reviews in Food Science and Nutrition,
53(11), 1191e1201.