Food Chemistry 228 (2017) 243248

Contents lists available at ScienceDirect

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

Effect of a multiple freeze-thaw process on structural and foaming

properties of individual egg white proteins
Xiang Duan a,1, Junyi Li a,1, Qinjun Zhang a, Tong Zhao a, Mei Li a, Xueming Xu 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: In this study, major albumen proteins (ovalbumin, ovomucoid, ovotransferrin, lysozyme and ovomucin)
Received 9 November 2016 were singly subjected to a multiple freeze-thaw process, and the resulting changes in structural charac-
Received in revised form 31 January 2017 teristics and foamability were investigated. Structural changes of proteins occurred during the process,
Accepted 1 February 2017
regarding by sulfhydryl-disulfide interchange and exposure of hydrophobic groups. The differential scan-
Available online 3 February 2017
ning calorimetry and scanning electron microscopy showed that these albumen proteins underwent
denaturation, dissociation and possibly aggregation. Correspondingly, the foaming ability of albumen
Keywords:
proteins improved after the freeze-thaw treatment, except for ovalbumin. The foaming ability of whole
Albumen proteins
Freeze-thaw
egg white was higher than that of each albumen protein, and improved after the multiple freeze-thaw
Structural characteristics process. This study extended knowledge of the relative contribution of each albumen protein to foaming
Foamability properties of whole egg white during a freeze-thaw process, and suggested that a multiple freeze-thaw
process is a promising technique for improving foaming properties of egg white proteins.
2017 Published by Elsevier Ltd.

1. Introduction proteins underwent a structural change during frozen storage

Based on its excellent foaming ability, egg white is widely used
in a variety of food products, including cakes, dessert shells and
pies. For these food products, foams provide unique and desirable
textures and largely affect the final quality. Thus, technologies that
can improve foaming properties of egg white are greatly desired
for food industry.
Foam comprised of millions of bubbles each encapsulated by a
protein film and separated by a thin water filled lamella. Egg white
foam is created as liquid egg whites are whipped. During this pro-
cess, air comes into solution to form bubbles, white proteins
adsorb rapidly at the air-water interface and undergo rapid confor-
mational changes to form cohesive viscoelastic films around bub-
bles. The spreading ability of a protein at the liquid surface
depends on the protein conformation. For albumen proteins, a cer-
tain degree of denaturation is benefit to their foam-forming capac-
ity (Campbell, Raikos, & Euston, 2003; Johnson & Zabik, 1981a).
Eggs are usually marketed as shell eggs. Due to the increase of
food industrys demand for eggs and egg products, egg white or
yolk in the form of liquid, frozen or dried are available for ease
transport and storage. Previous studies reported that egg white
Corresponding author.
E-mail address: xueboliu@nwsuaf.edu.cn (X. Liu).
1
These authors contributed equally.
because of denaturation (Mori, 1971; Wootton, Hong, & Thi,
1981). Therefore, we are interested in learning whether a multiple
freeze-thaw (F-T) treatment can be implemented to enhance foam-
ing properties of white proteins via modifying their structures.
Actually, Zhao, Dong, Li, Kong, and Liu (2015) recently reported
that soy proteins treated with multiple F-T cycles exhibited an
improved functional properties due to partial structural unfolding.
The effect of a F-T process on the functionalities of whole egg
white has been extensively studied, though results were conflict-
ing (Herald & Smith, 1989; Vaclavik & Christian, 2014; Wootton
et al., 1981; Xu, Shimoyamada, & Watanabe, 1997). Egg white con-
sists of a mixture of proteins, including ovalbumin, ovotransferrin,
ovomucoid, lysozyme, ovomucin and others (Mine, 2008). The
structure of egg white allows it to perform well in foams because
each albumen protein carries out a different function (Stadelman,
Newkirk, & Newby, 1995). Although the foaming properties of each
albumen protein has been adequately investigated, further
research on the functional behaviors of individual albumen pro-
teins during a multiple F-T process are little reported so far. Thus,
investigating the functional behaviors of individual albumen pro-
teins subjected to a multiple F-T process is essential to evidence
the relative contribution of each albumen protein to functionalities
of whole egg white. In this study, these individual albumen

http://dx.doi.org/10.1016/j.foodchem.2017.02.005
0308-8146/ 2017 Published by Elsevier Ltd.
244 X. Duan et al. / Food Chemistry 228 (2017) 243248
proteins were singly subjected to a multiple F-T treatment, and
their structural and foaming properties were determined.
The aim of present work was to elaborate on the effect of a mul-
tiple F-T treatment on foaming properties of individual albumen
proteins. The relationships between changes in structural proper-
ties and foaming properties of these proteins were elucidated by
determining free sulfhydryl groups (SH), surface hydrophobicity
(Ho), thermal property, morphology and foaming properties as a
function of F-T cycles.
2. Material and methods
2.1. Materials
Fresh hen eggs laid within 24 h were collected from a local farm
(Yangling, Shaanxi, China). Ovotransferrin (C0755), lysozyme
(62971) and the testing chemicals including 1-
anilinonaphthalene-8-sulfonic acid (ANS), 5,50 -dithiobis (2-
nitrobenzoic acid) (DTNB) were purchased from Sigma-Aldrich
Co. LLC. (Shanghai, China). All other reagents used were of analyt-
ical grade (Shanghai Chemical Reagents Co., Shanghai, China).
2.2. Preparation of individual albumen proteins
The fresh eggs were broken manually and white and yolk were
carefully separated. Ovalbumin was separated according to the
method of Geng et al. (2012). Briefly, the egg whites (270 g) were
stirred using a magnetic stirrer (IKA, IKA Works Inc., Wilmington,
NC, USA) at 4 C for 2 h with 3 volumes of 50 mM NaCl solution.
The pH of solution was adjusted to 6.0 with 2 M HCl, and PEG-
8000 was added while stirring (final wt/wt: 15%). The dispersion
was allowed to stand for 2 h at 4 C, and then was centrifuged at
15,000g at 4 C for 10 min. The supernatant (ovalbumin) was dia-
lyzed against Milli-Q water at 4 C for 72 h with changes of water
every 6 h, and then lyophilized. Ovomucoid was prepared with the
modified procedure of Lineweaver and Murray (1947). The egg
whites were slowly diluted with 1 vol of freshly 10% (wt/v) tri-
chloroacetic acid (pH 1.15) and stirred mildly. The pH was main-
tained at 3.5 by addition of 1 N NaOH or 1 N HCl. Following
standing for 4 h at 4 C, the dispersion was centrifuged at 3000g
at 4 C for 30 min. The supernatant was filtered through cellulose
filter paper (pore aperture 3050 lm) and diluted with 3 volumes
of cold acetone and stored overnight at 4 C. The suspension was
centrifuged at 3000g at 4 C for 10 min and the precipitate (ovomu-
coid) was dissolved in water (1:10, wt/v). The solution was dia-
lyzed against Milli-Q water at 4 C for 72 h with changes of
water every 6 h, and then lyophilized. Ovomucin was isolated
according to the method of Omana and Wu (2009). Briefly, the
egg whites were homogenized using a magnetic stirrer for
30 min at ambient temperature (22 C). Stirring speed was set at
350 rpm to avoid foaming. The dispersion was diluted with 0.1 N
NaCl to triple volume. The pH of dispersion was then adjusted to
6.0 using 1 N or 0.1 N HCl, while stirring gently at ambient temper-
ature (22 C) for 30 min. The dispersion was stored overnight at
4 C and separated by centrifugation at 15,000g for 10 min at
4 C. The precipitate (ovomucin) was freeze-dried and stored at
20 C until analysis. Rather than native proteins in fresh eggs,
these individual proteins extracted for analysis could be seen as
pre-denatured state.
2.3. F-T treatment
For hen eggs, protein portion constitutes about 10% of whole
egg white. Ovalbumin, ovomucoid, ovotransferrin, ovomucin and
lysozyme occupied 54%, 11%, 12%, 3.5% and 3.4%, respectively
(Mine, 2008). Therefore, the five proteins were redissolved sepa-
rately in 5.4% (wt/v), 1.1% (wt/v), 1.2% (wt/v), 0.35% (wt/v), and
0.34% (wt/v) in deionized water. These dispersions and fresh egg
whites were separately stored at 20 C for 24 h, followed by
thawing at 22 C for 12 h. The F-T process was repeated for 1, 3
and 5 times, respectively. These samples were then lyophilized
and stored at 20 C. The samples without the F-T treatment were
considered as untreated samples.
2.4. Free SH group content
The free SH group content was measured according to the
method of Li, Zhu, Zhou, and Peng (2012). Ellmans 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 ethylenedi-
aminetetraacetic acid (EDTA) in 1000 mL of deionized water, pH
8.0). The lyophilized protein samples 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 protein sample was mixed with
0.04 mL of Ellmans reagent solution. Subsequently, the mixture
was shaken and incubated in dark at room temperature for
10 min. The absorbance of supernatant was measured at 412 nm
against the blank (without Ellmans reagent and sample). Absor-
bance values were converted to amounts of free SH groups using
a calibration curve with reduced glutathione (Wang et al., 2014).
2.5. Surface hydrophobicity (Ho)
Ho of albumen proteins was determined by following the
method of Kato and Nakai (1980), with slight modifications. Each
sample was diluted to five concentrations between 0% and 0.1%
protein (wt/wt) in 10 mM phosphate buffer (pH 7.0). Then, aliquots
(20 lL) of ANS (8.0 mM in the same buffer) were added to 4 mL of
each protein solution. The solutions were rested for 3 min in dark
and placed in the cell of a Hitachi F4500 fluorescence spectrometer
(Tokyo, Japan), and the fluorescence intensity 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. Thermal analysis
Thermal properties of albumen proteins were analyzed by dif-
ferential scanning calorimetry (DSC). DSC was carried out accord-
ing to Wang et al. (2014) using a DSC Q2000 instrument (TA
Instruments, New Castle, DE, USA). The freeze-dried proteins (2
3 mg) were weighed in aluminum pans, the pans were hermeti-
cally sealed. A peltier device was used to heat samples from 20
to 180 C at 10 C/min, while gas nitrogen (50 mL/min) was used
to purge. An empty pan served as the reference. The enthalpy value
was calculated from the thermogram using the DSC Q2000 V24.2
Build 107.
2.7. Scanning electron microscope (SEM)
The morphology of albumen protein samples were observed
with a SEM. The lyophilized samples were sputter-coated with
gold, and examined in a Hitachi S-3400N scanning electron micro-
scope (Hitachi, Tokyo, Japan) at 5.0 kV.
2.8. Foaming properties
The foaming properties of each albumen protein was deter-
mined by the method of Akintayo, Oshodi, and Esuoso (1999), with
 X. Duan et al. / Food Chemistry 228 (2017) 243248
slight modifications. Briefly, weighed amounts of freeze-dried pro-
teins (100 mg) were dispersed in 10 mL distilled water. The result-
ing solutions were homogenized at a speed of 10,000 rpm, using a
homogenizer (IKA Labortechnik, Selangor, Malaysia) to incorporate
air for 2 min at room temperature. The whipped samples were
immediately transferred into a 25 mL cylinder and total volume
was read after 30 s. The foaming capacity was calculated according
to the following equation:
AB
Foaming capacity %  100
B
where A was the volume after whipping (mL), B was the volume
before whipping (mL).
The whipped samples were allowed to stand at 20 C for 30 min
and the volume of whipped samples were then recorded. Foaming
stability was calculated as follows:
CB
Foaming stability %  100
B
where C was the volume after standing (mL), B was the volume
before whipping (mL).
2.9. 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-
cans multiple-range tests were conducted to determine differ-
ences between the means. Results were considered statistically
significant at P < 0.05.
3. Results and discussion
3.1. Free SH group content
Disulfide bonds play a key role in maintaining structure of egg
proteins. Inter and/or intramolecular sulfhydryl-disulfide inter-
change reactions are involved in foam formation (Johnson &
Zabik, 1981a). The free SH contents in albumen proteins and
egg whites with different F-T cycles are presented in Fig. 1. For
ovalbumin and ovotransferrin, there was no significant difference
between the untreated and F-T treated groups. Among albumen
proteins, ovalbumin is the only protein that has free SH groups,
containing a single disulfide bond between Cys74 and Cys121.
Ovotransferrin contains 15 disulfide bridges which maintain its
bilobal structure (Mine, 2008). Free SH contents of the two pro-
teins were scarely influenced by a multiple F-T treatment, indicat-
ing that disulfide bonds of the both proteins were stable during the
process. Meanwhile, the free SH content in ovomucoid signifi-
cantly decreased from 0.92 lmol/g to 0.31, 0.50 and 0.29 lmol/g
after 1, 3 and 5 F-T cycles, respectively. The ovomucoid molecule
is composed of three distinct domains crosslinked only by intrado-
main disulfide bonds (Mine, 2008). The decrease in free SH con-
tent was due to the alteration in SS/SH exchange reaction or SH
oxidation and rearrangement (Chen et al., 2016). Contrarily, the
free SH content in lysozyme significantly increased from
0.28 lmol/g to 0.61, 0.70 and 0.45 lmol/g after 1, 3 and 5 F-T
cycles, respectively. Native lysozyme contains four disulfide bonds
(Johnson & Zabik, 1981a). Disulfide bonds are relatively weak cova-
lent bonds, they could be broken during a F-T process because of
water redistribution and ice recrystallization (Wang, Jin, & Xu,
2015; Zhao, Li, Liu, Liu, & Li, 2012). Thus, the increase in free SH
content of lysozyme was attributed to the depolymerization effect
of lysozyme through breakage of disulfide bonds during a F-T pro-
cess. Furthermore, the free SH content in ovomucin increased
245
Fig. 1. Free sulfhydryl (SH) group contents of untreated and treated albumen
proteins (1, 3 and 5 F-T cycles). Different letters (ac) in the same protein indicated
significant differences (P < 0.05).
when the number of F-T cycle increased from 0 to 3, and then
decreased to a value which was not significantly different from
the untreated sample. Ovomucin was considered as a biopolymer
which was cross-linked by disulfide bonds (Hayakawa & Sato,
1976). The result showed that few F-T cycles (1 and 3 F-T times)
could break these disulfide bonds and thus increased the SH con-
tent. Whereas, more F-T cycles (5 times) induced the new forma-
tion of disulfide bonds because of SH oxidation, which
decreased the free SH content in ovomucin. Similarly, the free
SH content in egg white increased significantly from 1 to 3 F-T
times, and then decreased after 5 F-T cycles to a value which
was lower than that of untreated sample. Howell and Taylor
(1995) proposed that the formation of disulfide bonds between
sulfhydryl groups enhanced foaming properties of globular pro-
teins. This meant that the foaming properties of egg white might
improve after a multiple F-T treatment.
3.2. Surface hydrophobicity (Ho)
Ho is a structural characteristic to evaluate conformational
change of proteins. The binding of fluorescent probe (ANS) to pro-
teins occurs at hydrophobic cavities formed by grouping of nonpo-
lar residues on protein surface, thus could give information on Ho
of proteins (Wang et al., 2014). Fig. 2 shows Ho values of albumen
proteins with different F-T cycles. For ovalbumin, a remarkable
decrease was observed in Ho value after a F-T treatment. This indi-
cated that ovalbumin folded or aggregated and thus the hydropho-
bic residues were buried in interior during a F-T process. The Ho
value of ovomucoid increased after 3 F-T cycles, and then
decreased to a value after 5 F-T cycles, which was still higher than
that of untreated sample. Combined with the free SH content
result, the increase in Ho value of ovomucoid after multiple F-T
cycles was originated from the molecular rearrangements, with
an exposure of hydrophobic side-chain groups. For ovotransferrin,
the Ho value fluctuated largely with increasing number of F-T
cycles. Ovotransferrin contains 15 disulfide bridges, and
Muralidhara and Hirose (2000) reported that the interdomain
disulfide bonds significantly decreased its conformational stability
in the C- lobe. Therefore, the less stability of structure might be a
reason that ovotransferrin underwent a large-scare conformational
transition during a multiple F-T process. Furthermore, the Ho value
246 X. Duan et al. / Food Chemistry 228 (2017) 243248

3.3. Thermal property

DSC analysis was carried out to investigate the effect of a F-T

Fig. 2. Surface hydrophobicity (Ho) of untreated and treated albumen proteins (1, 3
and 5 F-T cycles). Different letters (ac) in the same protein indicated significant
differences (P < 0.05).
of lysozyme increased from 205.35 to 234.56, 244.68 and 258.07
after 1, 3 and 5 F-T cycles. This trend was in agreement with the
free SH content result, suggesting that the increase in Ho value
was originated from the unfolded structure and thus the fluores-
cent aromatic amino acids transferred from core to surface. In
addition, Ho value of ovomucin increased from 700.50 to 823.75
after 1 F-T cycle, and then decreased to values which were lower
than that of untreated samples, reaching 402.83 and 107.57 after
3 and 5 F-T cycles, respectively. This suggested that ovomucin
molecules unfolded after 1 F-T cycle, and then began to aggregate
with increasing number of F-T cycles. Finally, it was notable that
the Ho value of egg white increased from 158.29 to 232.89,
241.02 and 238.05 after 1, 3 and 5 F-T cycles. The spontaneous
adsorption of proteins from solution to the air interface is of cen-
tral importance to their foaming properties (Wang, Tao, et al.,
2014). Hydrophobic patches on a proteins surface initially drive
this process, and surface hydrophobicity is correlated with
improved foaming properties (Townsend & Nakai, 1983). There-
fore, the increase in Ho after a F-T treatment enhanced amphipathy
of egg white and thus improved its foaming properties.
treatment on thermal property of albumen proteins. The denatura-
tion temperature (Tp) and enthalpy (DH) are main parameters of
DSC profile. Egg protein denaturation is one major importance in
establishing cake structure, thus alteration of albumen proteins
denaturation behavior may affect final properties of egg products.
Table 1 showed Tp of individual albumen proteins with different F-
T cycles. Ovalbumin is the most abundant protein in egg but is rel-
atively heat-labile (Mine, 2008). The Tp of untreated and F-T trea-
ted ovalbumin was below 60 C. This was lower than the data
reported by Donovan, Mapes, Davis, and Garibaldi (1975) indicat-
ing that Tp of ovalbumin was 84 C. We guessed that the difference
was because that the protein samples in this work were in freeze-
dried powder rather than protein solution. For ovalbumin and ovo-
transferrin, no significant difference in Tp was seen between
untreated and F-T treated samples. This suggested that the both
proteins maintained thermal stability during a F-T treatment. This
result was in agreement with the SH result showing that disulfide
bonds of the two proteins were stable during the process. For ovo-
mucoid, Tp increased from 112.06 C to around 120 C after a F-T
treatment. Combined with above-mentioned SH and Ho results,
the increase in heat stability of ovomucoid was contributed to
the enhanced intermolecular forces. Contrarily, Tp of lysozyme
decreased from 118.19 C to 112.42 C, 106.29 C and 102.11 C
after 1, 3 and 5 F-T cycles, respectively. This might be due to
breakup of disulfide bonds and less ordered structures. Moreover,
Tp of ovomucin fluctuated during a F-T process, and the Tp of 5 F-
T treated sample (116.80 C) was significant lower than that of
untreated sample (123.63 C). This was attributed to its flexible
structure (Donovan et al., 1975), which was easily affected by a
F-T process. Finally, it was notable that the Tp of F-T treated egg
white was lower than that of untreated sample (145.10 C), which
suggested that weaker protein-protein interactions and partly
denaturation occurred during the process.
The DH values obtained from the thermograms of DSC generally
reflect competition between endothermic unfolding and exother-
mic aggregation (Kato, Ibrahim, Watanabe, Honma, & Kobayashi,
1990). The DH of albumen proteins with different F-T cycles are
shown in Table 2. There was no significant change in DH of ovalbu-
min before and after a F-T treatment. This trend was in agreement
with the SH and Tp results. The DH values of ovomucoid
decreased significantly after 5 F-T cycles treatment. This suggested

Table 1
Denaturation temperature (Tp) of untreated and treated albumen proteins (1, 3 and 5 F-T cycles). Different letters (ac) in the same column indicated significant differences
(P < 0.05).

F-T cycles Denaturation temperature Tp (C)

Ovalbumin Ovomucoid Ovotransferrin Lysozyme Ovomucin Egg white
Untreated 58.91 0.85 112.06 1.02b 113.44 1.25 118.19 1.09a 123.63 1.01a 145.10 1.37a
1 59.51 0.78 119.40 1.32a 110.83 1.21 112.42 1.17b 120.67 0.94ab 120.93 0.55c
3 58.56 0.21 119.50 0.78a 114.68 1.30 106.29 0.63c 122.49 1.36a 130.49 1.20b
5 58.82 0.60 120.14 0.42a 115.24 1.14 102.11 1.45c 116.80 1.52b 128.05 1.26b

Table 2
Enthalpy of denaturation (DH) of untreated and treated albumen proteins (1, 3 and 5 F-T cycles). Different letters (ad) in the same column indicated significant differences
(P < 0.05).

F-T cycles Enthalpy of denaturation DH (J/g)

Ovalbumin Ovomucoid Ovotransferrin Lysozyme Ovomucin Egg white
Untreated 148.9 4.3 204.0 5.8a 181.1 4.2b 132.5 1.1a 127.2 3.1b 94.0 3.3d
1 154.6 2.7 204.0 3.9a 215.9 1.9a 131.5 3.0a 155.3 1.8a 198.6 2.8a
3 153.8 3.8 199.4 2.6a 158.4 4.0c 106.3 3.1b 52.7 1.6c 142.1 2.5c
5 152.2 2.4 185.7 2.2b 182.6 2.7b 69.1 1.6c 155.1 2.3a 161.0 3.0b
 X. Duan et al. / Food Chemistry 228 (2017) 243248 247

Fig. 3. SEM images (1.0 k  magnification) of untreated and treated albumen proteins (1, 3 and 5 F-T cycles).

that ovomucoid underwent a progressive denaturation after 5 F-T

treatment involving disruption of ordered structures (Wang, Xu,
et al., 2014). Similarly, the DH values of lysozyme decreased from
132.5 J/g to 106.3 and 69.1 J/g after 3 and 5 F-T cycles treatment,
which was in agreement with the Tp result showing that the heat
stability of lysozyme decreased with increasing F-T cycles. For ovo-
transferrin and ovomucin, the DH values fluctuated during the pro-
cess. Finally, the DH value of egg white increased from 94.0 J/g to
198.6, 142.1, and 161.0 after 1, 3 and 5 F-T cycles, suggesting that
egg white underwent an increasing level of structure folding or
denaturation during the process.

3.4. Morphology

To have tangible evidence of the transformation of albumen

proteins microstructure, we employed SEM (Fig. 3). The untreated
samples developed spongy structure and had large pores, owing to
recrystallization. After a multiple F-T treatment, these albumen
proteins collapsed into irregular shapes and aggregated into larger
rigid structures, which was due to the growth of ice crystal and
redistribution of water. This suggested that partial rearrangement
and denaturation and aggregation occurred during a F-T process.
Combined with above-mentioned results, the forces involved in
rearrangement and denaturation of albumen proteins included
disulfide bonds, hydrophobic interactions and others. Foaming
properties of proteins are generally influenced by surface flexibility
and hydrophobic properties. Furthermore, proteins with random
coil flexible structure have better foaming properties than highly
ordered structures for its faster absorption rate into air interfaces
(Graham & Phillips, 1979). Therefore, a multiple F-T treatment
could lead to partial denaturation of these molecules, which poten-
tially contributes to the improvement of foaming properties.

3.5. Foaming properties

In this work, foaming properties of albumen proteins are deter-
mined and shown in Fig. 4, the foaming properties of ovomucin
were not determined for its low solubility. For ovalbumin, it could
be seen that there was no significant difference in foaming ability
(FA) during a F-T process. This was in agreement with the SH and
DSC results indicating that ovalbumin maintained disulfide bonds
stability and thermal stability during a F-T process. Whereas the
foaming stability (FS) of ovalbumin decreased after 1 F-T cycle,
and then began to increase with increasing F-T cycles. For ovomu-
coid and ovotransferrin, the FA gradually increased with increasing
F-T cycles. This was attributed to the unfolded and rearranged con-
formation due to a F-T treatment, enhancing the ability to absorb
at air-water interfaces. Furthermore, the FA and FS of lysozyme
Fig. 4. Foaming ability (FA) and foaming stability (FS) of untreated and treated
albumen proteins (1, 3 and 5 F-T cycles). Different letters (ac) in the same protein
indicated significant differences (P < 0.05). The foaming properties of ovomucin
were not determined for its low solubility.
were lower than that of other proteins. This was in agreement with
previous study reporting that lysozyme exhibited low foaming
characteristics (Johnson & Zabik, 1981a). Meanwhile, a fluctuation
in FA and FS of lysozyme was seen during a multiple F-T treatment.
Finally, it could be seen that the FS values of egg white were higher
than that of other individual protein samples. This was because
that egg white contained ovomucin, ovoglobulin and other albu-
men proteins, which could complexed with each other via inter-
molecular interactions. For example, ovomucin was reported to
248 X. Duan et al. / Food Chemistry 228 (2017) 243248
serve physical functions such as maintaining structure and viscos-
ity of egg white albumen, which played a key role in improving FA
and FS of egg white and other albumen proteins (Johnson & Zabik,
1981a; Sunwoo & Gujral, 2015). Ovotransferrin also participates in
coagulation and modifies foaming properties of other albumen
proteins (Johnson & Zabik, 1981b). Therefore, albumen proteins
may not behave similarly in isolation and in mixture with other
egg white proteins. For whole egg white, the FA increased from
27.4% to 31.2%, 33.5% and 35.4%, while the FS increased from
24.1% to 28.0%, 31.7% and 32.6% after 1, 3 and 5 F-T cycles, respec-
tively. The improvement in foaming properties was partially attrib-
uted to the increased Ho values, as the exposure of hydrophobic
groups could result in increased interactions at air-water interface
(Damodaran, 1994). In addition, the spontaneous adsorption of
proteins from solution to air-aqueous interface is key to their
foaming performance (Graham & Phillips, 1979), the above-
mentioned variations of denaturation/aggregation of albumen pro-
teins are an indicator of enhancement of foaming properties.
Above results indicated that application of a multiple F-T pro-
cess led to albumen proteins unfolding and exposure of hydropho-
bic groups which are essential for adsorption of protein onto the
air-water interfaces (Jambrak, Lelas, Mason, Kreic, & Badanjak,
2009). However, in egg products, the situation is complicated by
the presence of other species such as fat or sucrose which are
known to influence the foaming properties. Therefore, the effects
of interactive forces among ingredients on foaming properties of
albumen proteins during a F-T process will be further evaluated.
4. Conclusion
In this work, the structural and foaming properties of individual
albumen proteins during a F-T process were investigated. The
results indicated that a multiple F-T process could modify the
molecular arrangements and interactions of albumen proteins via
varifying their sulfhydryl-disulfide interchange and surface
hydrophobicity. These structural modifications resulted in protein
denaturation, dissociation and possibly aggregation, which were
reflected by changes in morphology and thermal properties. In par-
allel, a multiple F-T process enhanced foaming properties of some
individual albumen proteins and whole egg white. However, egg
white contains a wide array of other components, including trace
lipid, carbohydrate and minerals. Exploring the influence of these
components on functional properties of each albumen protein
and their potential interactions during a multiple F-T process is
also valuable.
Acknowledgment
This work was supported by grants from the National Natural
Science Foundation of China (Nos. 31501426 and 31671859).
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