Food Chemistry 252 (2018) 181–188

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

Emulsion stability and dilatational viscoelasticity of ovalbumin/chitosan T

complexes at the oil-in-water interface
⁎
Wenfei Xionga,b, Cong Rene, Mo Tiana, Xuejun Yanga, Jing Lia,b, Bin Lia,b,c,d,
a
College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China
b
Key Laboratory of Environment Correlative Dietology (Huazhong Agricultural University), Ministry of Education, China
c
Hubei Collaborative Innovation Centre for Industrial Fermentation, Hubei University of Technology, Wuhan 430068, China
d
Functional Food Engineering & Technology Research Center of Hubei Province, Wuhan 430068, China
e
Department of Basic Course Teaching and Research, Henan University of Animal Husbandry and Economy, Zhengzhou 450011, Henan, China

A R T I C L E I N F O A B S T R A C T

Keywords: The contribution of the emulsion rheological properties and the viscoelastic of the interface adsorbed layer to the
Electrostatic interactions emulsification mechanism of ovalbumin (OVA)-chitosan (CS) mixtures were investigated. In comparison to the
Gel-like emulsion treatment with OVA alone and OVA/CS mixtures at pH 4.0, the addition of CS at pH 5.5 increased the size
Viscoelastic properties distribution of emulsion droplets with significant flocculation through polyelectrolyte bridging, remarkably
Interfacial rheological properties
enhancing the emulsions stability against gravity creaming after storage at 25 °C for 14 days. The dynamic
rheological properties indicated that the formation of the complex at pH 5.5 increased the elastic modulus (G′)
and apparent viscosity (η∗) of the emulsions, which is useful for inhibiting creaming. Moreover, the com-
plexation of OVA and CS at pH 5.5 increased the dilatational modulus (E), especially the elastic modulus (Ed), of
the oil/water interfacial absorbed layer, which could reduce the droplet coalescence and therefore inhibit the
growth of emulsion droplets.

1. Introduction surface-active (Dickinson, 2003). The addition of polysaccharides can

Proteins are commonly applied in food industry as emulsifying
agents. However, emulsions prepared with proteins alone are typically
unstable against aggregation around the isoelectric point of proteins
due to weakened electrostatic repulsion (Evans, Ratcliffe, & Williams,
2013). Polysaccharides can be used to stabilize emulsions by adsorbing
on emulsion droplets therefore controlling colloidal interactions, in-
creasing viscosity to reduce droplet aggregation, or creating a yield
stress to rest particle motion (Ganzevles, Stuart, van Vliet, & de Jongh,
2006). Additionally, because of co-soluble mixtures, soluble complexes
and coacervates between proteins and polysaccharides could be formed
at specific pH values via electrostatic interactions in aqueous solutions
(Turgeon, Beaulieu, Schmitt, & Sanchez, 2003). Consequently, the
overall stability of emulsions stabilized with proteins and poly-
saccharides depends not only on the functional properties of the in-
dividual ingredients, but also on their interactions (Zinoviadou,
Scholten, Moschakis, & Biliaderis, 2012). For this reason, such com-
plexes or coacervates of protein/polysaccharide generated by electro-
static interactions have received considerable interest in recent years to
stabilize oil-in-water (o/w) emulsions (Ratcliffe & Williams, 2013).
Polysaccharides are predominantly hydrophilic and thereby are not
obviously modify the rheology behaviour of the bulk phase of emul-
sions, and it also plays a critical role for the protein adsorption from the
bulk phase to the interface (Murray, 2002). Generally, protein ad-
sorption on the oil-in-water interface mainly includes three stages: (i)
diffusion from the bulk to the interface, (ii) actual adsorption at the
interface, and (iii) conformational reorganization at the interface
(Dickinson, 2011). The diffusion of protein from the bulk to the inter-
face is generally determined by the protein concentration in the con-
tinuous phase, but this is not usually the predominant or rate-limiting
stage in protein solutions with low concentrations (Dickinson, 2011).
For the protein/polysaccharide mixtures, when protein and charged
polysaccharide are introduced simultaneously, the steps of diffusion,
adsorption, and reorganization can be affected at different extents due
to the electrostatic interactions between the two biopolymers
(Dickinson, 2008). It was reported that the interactions between NaCas
and xanthan gum (XG) played a significant role in the dynamic char-
acteristics of NaCas adsorbed layers, which increased the diffusion,
penetration and rearrangement rate of NaCas at the oil-in-water inter-
face (Liu, Zhao, Liu, & Zhao, 2011; Liu, Zhao, Zhou, & Zhao, 2016; Liu
et al., 2012). Nevertheless, the availability of the hydrophobic binding
sites on the protein could be reduced due to the interactions between

⁎
Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
E-mail address: libinfood@mail.hzau.edu.cn (B. Li).

https://doi.org/10.1016/j.foodchem.2018.01.067
Received 13 September 2017; Received in revised form 27 November 2017; Accepted 8 January 2018
Available online 09 January 2018
0308-8146/ © 2018 Elsevier Ltd. All rights reserved.
W. Xiong et al.
ovalbumin and sulphated polysaccharide, leading to a lower surface
activity (Galazka, Dickinson, & Ledward, 2000). The opposite results
may be ascribed to various physiochemical properties of proteins and
polysaccharides.
Therefore, for the protein/polysaccharide mixtures, research on the
adsorption behaviour of proteins and the interfacial rheology properties
of adsorbed layers can provide a useful insight into the mechanistic
origin of some emulsion properties (Jourdain, Schmitt, Leser, Murray, &
Dickinson, 2009). Ovalbumin (OVA) is the main protein in egg white,
and is commonly used as emulsifier in food industry. Previous studies
showed that the hydrophobicity and net charge of OVA had important
effects on its interfacial adsorption properties (Croguennec, Renault,
Beaufils, Dubois, & Pezennec, 2007; Delahaije, Wierenga, van
Nieuwenhuijzen, Giuseppin, & Gruppen, 2013; Wierenga, Meinders,
Egmond, Voragen, & de Jongh, 2003, 2005), and OVA/anion poly-
saccharide complex coacervates showed a better emulsifying ability
compared with OVA alone (Galazka et al., 2000; Niu et al., 2015). In
our past work, OVA and chitosan (CS) could form a co-soluble mixture
and complexes at pH4.0 and pH5.5, respectively (Xiong et al., 2016).
However, the effect of the electrostatic interaction between OVA and CS
on the adsorption behaviour of protein at oil-in-water interface and the
viscoelastic properties of absorbed layer, and the relationship between
this effect and emulsion stability has not been reported. The aim of this
work was to explore the effect of OVA-CS interaction on the emulsion
stability, and reveal the stabilized mechanism of emulsions prepared by
OVA-CS mixtures at different pH values in the bulk phase. Furthermore,
the interfacial dilatational properties of OVA/CS absorbed layers at oil-
in-water interface were also assessed by the interface rheological
measurements.
2. Materials and methods
2.1. Materials
Ovalbumin (OVA, A5503, > 98% pure by agarose electrophoresis)
was purchased from Sigma Co. (St. Louis, MO) without further pur-
ification. Chitosan (CS, deacetylation degree about 90.5%, and the
molecular weight about 350 kDa.) was purchased from Qingdao
Yunzhou Biochemistry Co., Ltd. (Shandong, China). Soybean oil was
purchased from the local supermarket. Other reagents were obtained
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All
reagents were analytical grade unless otherwise stated.
2.2. Preparation of OVA/ CS aqueous complexes
OVA (1%, w/v) stock solution was obtained by dissolving OVA
powder in deionized water under gentle stirring at 25 °C for 2 h and
then stood overnight at 4 °C to ensure a complete protein hydration.
Moreover, sodium azide (0.02%, w/v) was added before stirring to
inhibit bacteria growth. CS (1%, w/v) stock solution was prepared by
dissolving CS powder in acetic acid solution (1%, w/v) and stirring at
25 °C overnight. OVA/CS (OVA:CS = 3:1, w/w) mixture was fabricated
by mixing corresponding stock solutions and stirring for 30 min, and
then the pH values of OVA alone solution and OVA/CS mixed solution
were adjusted to 4.0 and 5.5 using 0.5 M HCl and 0.5 M NaOH. The
total biopolymer concentration was fixed at 0.3% (w/w) for both OVA/
CS mixtures and individual OVA.
2.3. Emulsions preparation
Primary oil-in-water emulsions (100 ml) were prepared by mixing
the soybean oil (20 ml) and solution of OVA alone or OVA/CS mixtures
(80 ml) using a high-speed homogenizer (IKA-ULTRA-TURRAX T25
Wilmington, NC) operating at 10,000 rpm for 1 min. The oil fraction
was fixed at 20% (w/w). For all emulsions, the total biopolymer was
fixed at 0.3% (w/w) without salt ion. Furthermore, the primary
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Food Chemistry 252 (2018) 181–188
emulsions were further processed with two passes through a high-
pressure homogenizer (AH100B, ATS Engineering Inc. Canada) at
30 MPa. These freshly prepared emulsions were directly subjected to
analysis or storage stability experiments at 25 °C.
2.4. Particle size measurement of emulsions
The volume-average droplet size distribution (d4,3) of freshly pre-
pared emulsions at pH 4.0 and 5.5 were measured by using Malvern
MasterSizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire,
U.K.). Before the test, all the emulsions were diluted 100 times using
phosphate buffer solutions (PBS, 5 mM) with corresponding pH values,
the pH values of PBS were adjusted to 4.0 and 5.5 using 0.5 M HCl and
0.5 M NaOH. The refractive index of the emulsions, dispersed phase and
continuous phase was set to 1.095, 1.456 and 1.333, respectively. All
measurements were conducted at least in duplicate.
2.5. Optical microscopy
The morphology of emulsions was studied using Ningbo shunyu CX
40 optical microscope (China) and the images were captured with a
OD1400Y digital camera (China).The emulsions were diluted 100 times
using PBS solutions (5 mM) with corresponding pH values, and then
placed on a glass slide with a cover glass for microscopic observation at
25 °C.
2.6. Creaming index of emulsions
The creaming stability of emulsions with different formulations at
various pH values was evaluated visually upon quiescent storage for up
to 14 days. Each emulsion was filled in a glass test tube and then per-
pendicularly stored at 25 °C. The height of the serum (Hs, cm) and total
height of the whole fresh emulsion (Ht, cm) were recorded at various
periods of storage. The creaming index was calculated using Eq. (1):
Creaming index (CI, %) = (Hs /Ht) × 100 (1)
2.7. Rheological measurement of emulsions
The dynamic rheological properties and steady shear viscosities (η)
of emulsions were performed using a strain-controlled rheometer
(AR2000ex, TA Instruments, USA) fitted with parallel plate geometries
(40 mm in diameter). Fresh emulsions were loaded and maintained on
the plate for 10 min to achieve thermal equilibrium. The gap value
between two plates was set to 1.0 mm. The storage modulus (G′) and
loss modulus (G″) of emulsions were measured with varying the fre-
quency from 0.1 to 100 rad/s. Strain sweep tests were conducted to
determine the proper conditions of rheological measurements, and the
strain of all samples was set to 0.5%. The temperature of the rheological
experiment was set at 25 °C. Meanwhile, the viscosity of the emulsions
was recorded as the shear rate shifted from 0.1 to 100 1/s.
2.8. Dynamic interfacial surface pressure (π) and interface viscoelasticity
measurement
2.8.1. Measurement of interfacial surface pressure
The interfacial surface pressure (π) and adsorption kinetics of OVA
alone or OVA/CS mixtures at the oil/water interface were performed
using an automated drop tensiometer (Tracker-H, Teclis, France) at
25 °C. For this test, to eliminate the interference of impurities in com-
mercial soybean oil, and thus to obtain a clearer description of ad-
sorption and diffusion behaviour of the protein molecules at the oil-in-
water interface, medium chain fatty acid (MCT) was used instead of
commercial soybean oil for the study of interfacial rheological prop-
erties. The bulk phase (OVA or OVA/CS mixtures) and oil phase were
placed in the cuvette and syringe, respectively. The total biopolymer
W. Xiong et al.
concentration was fixed at 0.3% (w/w), and the test model was raising
hanging drop. Before test, dispersions and oil were allowed to stand for
at least 1 h to reach 25 °C. The temperature of the system was main-
tained constant by circulating water from a thermostat. The oil droplet
volume was 10 µl during the test. The oil/water interface pressure (π,
mN/m) was calculated with Eq. (2):
π (mN/m) = γ0−γ (2)
where γ0 (mN/m) and γ (mN/m) is the interfacial tension of pure oil-
distill water (26 mN/m) and OVA alone or OVA/CS mixed solutions,
respectively. The surface tension was determined by drop shape ana-
lysis (Benjamins, Cagna, & Lucassen-Reynders, 1996). All measure-
ments were performed up to 3 h.
2.8.2. Measurement of interfacial viscoelasticity
To obtain surface dilatational parameters, the dynamic interface
viscoelasticity of OVA alone and OVA/CS mixtures at the oil–water
interface were investigated using an automated drop tensiometer
(Tracker-H, Teclis, France) at 25 °C. OVA alone or OVA/CS mixtures
and oil were placed in the cuvette and syringe, respectively. The total
biopolymer concentration was fixed at 0.3% (w/w). The sinusoidal
interfacial compression and expansion were performed by changing the
drop volume at 10% of deformation amplitude within the linear regime,
and the oscillation frequency was 0.1 Hz, the droplets volume was
10 µl, and the experiment was started after equilibration 60 s. The
number of active and blanks was 5 cycles during the experiments.
Details of this experiment were described by Perez, Carrara, Sánchez,
Santiago, and Patino (2009).
2.9. Statistical analysis
Unless otherwise stated, all measurements were carried out in tri-
plicate. One way analysis of variance (ANOVA) with a 95% confidence
interval was used to compare the significance of the results obtained.
Statistical analysis was performed using SPSS software version 19.0.
3. Results and discussion
3.1. Microstructure and oil droplets size of emulsions
The microstructure, average oil droplets size distribution (d4, 3) of
fresh emulsions stabilized by OVA alone and OVA/CS mixtures are
presented in Fig. 1. From our previous study, the isoelectric point (pI) of
OVA was 4.85 (Xiong et al., 2016), which indicated that pH 5.5 was
much closer to pI of OVA than pH 4.0. Therefore, the electrostatic re-
pulsion between the droplets was insufficient to prevent flocculation at
pH 5.5 (Fig. 1B). Additionally, compared to the OVA stabilized emul-
sions, the incorporation of CS caused a wider size distribution of
emulsion droplets, and resulted in the increased droplets size from 21.2
to 33.8 µm (d4, 3) at pH 5.5 (Fig. 1D).
However, optical microscopy results showed extensive flocculation
of emulsion droplets in the presence of CS at pH 5.5. Indeed, OVA was
negatively charged while CS was positively charged at pH5.5, and ul-
timately forming the complexes with positive charge by electrostatic
attraction (Xiong et al., 2016). On the other hand, our past work sug-
gested that the optimum mass ratio of OVA/CS was 3.6:1 at pH 5.5 by
the isothermal titration calorimetry (ITC) (Xiong et al., 2016). This
indicated that the surface of emulsion droplets was not completely
coated with CS, which led to the occurrence of bridging flocculation
(Dickinson, 2008). At pH4.0, both OVA and CS were positively charged,
and no aggregation were observed due to the sufficient electrostatic
repulsion and steric hindrance.
3.2. Rheological properties of emulsions
To further investigate the macroscopic characteristics of the
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Food Chemistry 252 (2018) 181–188
emulsions, we assessed the dynamic viscoelastic properties and flow
behaviour of the fresh emulsions. Generally, the storage modulus re-
lates to the elasticity of emulsion, which reflects solid properties, and
loss modulus relates to viscidity of the emulsion, which represents li-
quid properties. As presented in Fig. 2A and B, the storage modulus (G′)
was considerably higher than loss modulus (G″) in the frequency sweep
range from 0.1 to 100 rad/s (except OVA/CS mixtures at pH 4.0),
suggesting the formation of predominantly elastic gel-like emulsions.
Specially, the G′ value of emulsion stabilized by OVA/CS complex at pH
5.5 was nearly 10 times greater than the other emulsions, and the G′
was almost independent on the sweep frequency in the test range of
0.1–10 rad/s. Additionally, it was worthy to note that the G′ of emul-
sion stabilized by pure OVA at pH 5.5 was slightly higher than pH 4.0,
which might be attribute to the higher aggregation degree of protein at
pH5.5 than pH 4.0 (Benichou, Aserin, & Garti, 2002; Dickinson, 1998;
Dickinson & Pawlowsky, 1997). These results were consistent with the
creaming stability of emulsion, and suggesting that higher G′ could
provide great creaming stability due to the droplet flocculation.
In addition, apparent viscosities of fresh emulsions as a function of
shear rate (0.1–100 1/s) in the absence or presence of CS at pH 4.0 and
pH 5.5 were illustrated in Fig. 2 C. As expected, all fresh emulsions
showed pseudo plastic flow behaviour in the whole shear rate range,
indicating deflocculation of associated droplets in the emulsions (Liu &
Tang, 2014). On the other hand, with the increase of shear rate, the
emulsion droplets and polymer chains became more ordered along the
flowing direction, which reduced the resistance to flow observed as a
lower viscosity. However, significant differences were still existed
among the emulsions with different formulations. Adding CS resulted in
a higher viscosity of emulsions compared with that stabilized by pure
OVA. In the case of emulsions without CS, the viscosity at pH 5.5 was
higher than that at pH 4.0, which may be related to the flocculation of
emulsion droplets. Specially, the emulsion stabilized by the OVA/CS
complex at pH 5.5 had the highest apparent viscosity, especially at
relatively low shear rate of 0.1–3 1/s, which greatly retarded the mo-
bility of the emulsion droplets and thus inhibited the creaming of
emulsions. These results were consistent with the emulsions stabilized
by sodium caseinate/flaxseed gum mixtures (Zhao et al., 2015). These
findings indicated that the flocculation could weaken the flow of
emulsions by resistance force among interparticles, thereby increasing
the apparent viscosity of emulsions.
3.3. Creaming stability
Fig. 3 showed the effect of CS addition on the creaming stability of
emulsions at pH 4.0 and pH 5.5. There was obvious difference in the
plots between the OVA stabilized emulsions with CS at pH 5.5 and the
OVA stabilized emulsions with or without CS at pH 4.0. In the absence
of CS, the creaming layer was soon observed, which might be attribute
to the low net charge of OVA (data not show). However, the creaming
index (CI, %) of OVA stabilized emulsion at pH 4.0 was higher than that
at pH 5.5. This result might be due to the fact that pH 5.5 was closer to
the isoelectric point of OVA, and the aggregation degree of protein
molecules was higher than pH 4.0, resulting in greater steric hindrance
and improving the stability of the emulsion. What’s more, significant
creaming behaviour was also observed at pH 4.0 for the emulsion in the
presence of CS, and the CI value was slightly greater than the emulsion
without CS at pH 5.5. Specifically, in the case of adding CS at pH 5.5, it
was surprisingly observed that no creaming occurred during the whole
storage up to 14 days, indicating extraordinary stability against
creaming, although the flocculation of emulsion was observed from the
microstructure and droplet size distribution as shown in Fig. 1D. In-
deed, the addition of CS increased the viscosity of the bulk phase and
effectively hindered the free movement of the emulsion droplets
(Fig. 3). On the other hand, the formation of the gel-like emulsion due
to the electrostatic cross-linking between OVA and CS provided a better
viscoelastic property at pH 5.5 (Fig. 2). This correlates well with the
W. Xiong et al.
Fig. 1. Optical micrographs of OVA/CS mixtures and pure OVA stabilized emulsions (20% soybean oil, 0.3% total biopolymer) at different pH values. A and B is pure OVA at pH 4.0 and
pH 5.5, respectively. C and D is OVA/CS complex at pH 4.0 and pH 5.5, respectively. Particle-size distributions of diluted emulsions are superimposed on the micrographs. The bar scale is
20 µm.
rheological results and is fully consistent with the degree of emulsion
flocculation. When polysaccharide concentrations well below satura-
tion coverage, the formation of polymer-cross-linked droplets network
induced by bridging flocculation makes the emulsions have a gel-like
rheological property, thus exhibiting excellent stability (Cao,
Dickinson, & Wedlock, 1990).
Moreover, the reversed emulsions after storage for 14 days still had
a great stability as observed from the inserted digital photos in Fig. 3.
This finding suggested that a gel-like network structure of emulsions
was formed involving extensive droplet flocculation, and thereby pro-
viding extraordinary creaming stability for the emulsion stabilized by
OVA/CS complex at pH 5.5. This phenomenon was also observed in the
emulsions stabilized by preheated soy protein (Liu & Tang, 2014; Tang
& Liu, 2013) or whey protein (Liu & Tang, 2011), and chitin Nano
crystal particles (Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis,
2011).
3.4. Interfacial adsorption and dilatational rheological properties
3.4.1. Adsorption kinetics and structural rearrangements at the oil-in-water
interface
As mentioned above, dynamic adsorption behaviour of protein
molecules in the oil-in-water interface in turn mainly involved
184
Food Chemistry 252 (2018) 181–188
diffusion, actual adsorption (penetration and unfolding) and con-
formational reorganization (Dickinson, 2011). Generally, there is a
rate-determining step, showing relatively low interfacial surface pres-
sures during the first step (Camino, Pérez, Sanchez, Patino, & Pilosof,
2009). The change in interfacial surface pressure (π) with adsorption
time (t) can be correlated by a modified form of Wardand Tordai
equation (Ward & Tordai, 1946):
π = 2C0 KB T (D t/3.14)1/2 (3)
where C0 is the concentration in the continuous phase, KB is the
Boltzmann constant, T is the absolute temperature, and D is the diffu-
sion coefficient. If the adsorption process is controlled by the protein
diffusion, a plot of π versus t1/2 will then be linear, and the slope of this
plot will be the diffusion rate (Kdiff) (MacRitchie, 1978), as shown in
Fig. 4A.
Furthermore, the first-order equation can be used to analyze the
rates of penetration and rearrangement of adsorbed protein molecules
at the interface (Graham & Phillips, 1979):
ln(πf −πt )/(πf −π0) = −k i t (4)
Where πf, π0, and πt are the interfacial pressures at the final ad-
sorption time, at the initial time, and at any time of each stage, re-
spectively, and ki is the first-order rate constant. The application of Eq.
W. Xiong et al. Food Chemistry 252 (2018) 181–188

Fig. 3. Creaming index (CI) as a storage time function for O/W emulsions.

Fig. 2. (A) The storage modulus (G′), (B) loss modulus (G″) and (C) apparent viscosity (η*)
of the fresh emulsions stabilized by the pure OVA and OVA/CS mixtures at pH 4.0 and pH
5.5, respectively. The experiments were performed at a constant frequency of 1 Hz or a
Fig. 4. (A) Time dependence of surface pressure (π) for OVA and OVA/CS adsorbed films
constant strain of 10%.
at the oil–water interface. Kdiff represent diffusion rate. (B) Typical profile of the mole-
cular penetration and configurational rearrangement steps at the oil–water interface for
(4) to the adsorption of the pure OVA and OVA/CS mixture at the in- OVA and OVA/CS mixed systems. Kp and Kr represent first-order rate constants of pe-
netration and rearrangement, respectively. The total biopolymer concentrations were
terface are presented in Fig. 5B. Clearly, there are two linear regions in
fixed at 0.3% (w/w, OVA: CS = 3:1).
these plots. Generally, the first slope is usually regarded as a first-order
rate constant of penetration (KP), while the second slope takes to a first-
order rate constant of molecular reorganization (KR) (Perez et al., penetration and reorganization at the oil-in-water interface were cal-
2009). culated and summarized in Table 1, the equilibrium interfacial pressure
According to Eqs. (3) and (4), the rate constants of protein diffusion, (π10800) was also included. In all cases, the interfacial surface pressure

185
W. Xiong et al. Food Chemistry 252 (2018) 181–188

2015).
Additionally, The Kdiff, Kp and Kr of the OVA alone solution at pH
5.5 were lower than at pH 4.0, which might be due to the poor solu-
bility caused by the limited diffusion of protein when pH was close to
pI. For the OVA/CS mixed system, the Kdiff at pH 4.0 (0.3260 mN/m/s1/
2
) was higher than at pH 5.5 (0.3010 mN/m/s1/2), possibly owing to the
greater hydrodynamic radius of the OVA/CS complex formed by elec-
trostatic attraction at pH 5.5, and then decreased the protein diffusion
(Ganzevles, Zinoviadou, van Vliet, Cohen Stuart, & de Jongh, 2006;
Ganzevles, Stuart, et al., 2006). Similar phenomena were also observed
in the sodium caseinate/xanthan gum mixtures system at the oil-in-
water interface (Liu et al., 2011) and β-lactoglobulin/pectin complexes
at the air–water interface (Ganzevles, Stuart, et al., 2006). Meanwhile,
compared with pure OVA system, the incorporation of CS decreased the
Kp and Kr, especially at pH 5.5. This phenomenon could be explained by
the fact that the formation of OVA/CS complex at pH 5.5 increased
molecular entanglement, and hindered the penetration and re-
organization of OVA molecules (Perez et al., 2009; Zhao et al., 2015).
On the other hand, the existence of a kinetic barrier associated with CS
adsorption could also affect the penetration of OVA (Jourdain et al.,
2009).

3.4.2. Dilatational rheological properties at the oil-in-water interface

Fig. 5. (A) Surface dilatational modulus (E) as a function of surface pressure (π) for OVA
and OVA/CS mixed systems at the oil–water interface. (B) Time-dependent dilatational
elasticity (Ed) for OVA and OVA/CS mixed systems adsorbed layers at the oil–water in-
terface. The total biopolymer concentrations were fixed at 0.3% (w/w, OVA: CS = 3:1).
Frequency, 0.1 Hz. Amplitude of compression/expansion cycle, 10%.
decreased with adsorption time (Fig. 4A), which could be related to the
protein adsorption at the interface (Perez et al., 2009; Wan et al., 2014).
Obviously, the addition of CS significantly decreased the equilibrium
interfacial pressure after adsorption for 180 min, especially at pH 5.5
(Table 1). As expected, there were much more protein molecules ab-
sorbed at the oil-in-water interface in the aqueous phase without CS,
resulting in a lower interfacial surface pressure. On the other hand, the
OVA/CS mixtures had a lower equilibrium interfacial surface pressure
at pH 5.5 compared with that at pH 4.0. It could be indicated that
electrostatic attraction between protein and polysaccharide resulted in
a lower surface activity through reducing the availability of the hy-
drophobic binding sites on the protein (Galazka et al., 2000). Similar
results have been found for sodium caseinate/xanthan gum system (Liu
et al., 2011, 2012) and sodium caseinate/flaxseed gum (Zhao et al.,
In general, the viscoelastic properties of the protein adsorbed layer
at air-in-water and oil-in-water interfaces can be the prediction of
foams and emulsions stability (Murray, 2002). Therefore, the surface
dilatational modulus (E) can reflect the mechanical strength of the
protein interfacial absorbed layer, which derives from the change in
interfacial tension (γ) (dilatational stress) resulting from a small change
in surface area (dilatational strain) (Lucassen & van den Tempel, 1972).
The surface dilatational modulus (E, E = Ed + iEv) includes real (sto-
rage component, Ed = |E|cosδ) and imaginary parts (loss component,
Ev = |E|sinδ), in which the phase angle (δ) between stress and strain is
a representation of the relative the viscoelasticity of interfacial ab-
sorbed layer (Rodríguez Patino, Rodríguez Niño, & Carrera Sánchez,
1999).
The evaluation of surface dilatational modulus (E) with interfacial
surface pressure (π) in the surface layer for the adsorption of pure OVA
and OVA/CS mixtures is presented in Fig. 5A. The E values were in-
creased immediately due to the adsorption of protein at the interface in
all cases. This result indicated that interaction between adsorbed pro-
tein molecules occurred (Wang et al., 2012). On the other hand, the
slopes of the E versus π curves were higher than 1.0, except in the
mixed system of OVA/CS at pH 4.0. These results reflected a non-ideal
behaviour for stronger molecular interactions between the film-forming
components of protein and polysaccharide, not just between the
amount of protein molecules adsorbed at the oil-in-water interface
(Camino, Sanchez, Rodríguez Patino, & Pilosof, 2011). Similar results
were also observed in other protein-polysaccharide systems (Liu et al.,
2011, 2016; Perez et al., 2009). However, for the pure OVA systems, the
slopes of E-π plots were higher than that in the OVA/CS mixed systems.
This finding might be related to the bulk protein concentration, which
was consistent with the diffuse of protein molecules (data not show).
Interestingly, the addition of CS increased the E values of the protein

Table 1
Characteristic parameters, including the apparent diffusion rate (Kdiff), constants of penetration and structural rearrangement at the interface (KP and KR), and surface pressure at the end
of adsorption (10,800 s, π10800) for OVA and OVA/CS mixed systems at pH 4.0 and pH 5.5.

Kdiff (mN/m/s1/2) (LR) Kp × 104 (LR) Kr × 104 (LR) π10800 (mN/m)

OVA (pH4.0) 0.35 ± 0.012 (0.9280)a

−3.5 ± 0.02 (0.9033)a
−23 ± 0 (0.9341)a
21 ± 1a
OVA (pH5.5) 0.34 ± 0.003 (0.9681)a −3.2 ± 0.02 (0.9549)b −22 ± 0 (0.9665)a 21 ± 1a
OVA/CS (pH4.0) 0.33 ± 0.006 (0.9525)b −2.6 ± 0.02 (0.8528)c −20 ± 0 (0.9776)b 20 ± 0a
OVA/CS (pH5.5) 0.30 ± 0.009 (0.9675)c −2.0 ± 0.03 (0.9510)d −14 ± 1 (0.9307)c 19 ± 0b

Different letters within the same column are significant (p < .05, n = 3) by Duncan’s multiple range test. LR is an abbreviation for linear regression coefficients.

186
W. Xiong et al.
interfacial absorbed layer, especially at pH 5.5, even though OVA
concentration was lower than the pure OVA systems. This phenomenon
implied that the interaction of electrostatic attraction between protein
and polysaccharide could improve the mechanical strength of oil –
water interfacial absorbed layer. This interfacial property was posi-
tively reflected in the emulsion stabilized by OVA/CS mixture at pH
5.5, and exhibited a fine emulsion stability.
Furthermore, the dynamic dilatational elastic modulus (Ed) of in-
terfacial layers of pure OVA and OVA/CS mixtures were presented in
Fig. 5B. Clearly, the Ed values were gradually increased due to the
adsorption of protein at the interface. After adsorption for 180 min, the
Ed values were close to the surface dilatational modulus (E values), and
the dilatational viscosity values were low (data not show). This result
indicated that the interface absorbed layer mainly exhibited the elastic
behaviour. Compared to the pure OVA systems, the presence of CS in-
creased the Ed values after adsorption for 180 min, especially at pH 5.5.
These findings suggested that the dilatational property of protein ab-
sorbed layer could be enhanced by protein-polysaccharide interactions.
Similar results were also observed for the NaCas/CMC mixtures (Liu
et al., 2016). In addition, the associative interactions between the
protein and polysaccharide increased entanglements (Xiong et al.,
2016), and then had a significant effect on the molecular and/or con-
densation of absorbed protein at the oil-in-water interface (Jourdain
et al., 2009).
In summary, the addition of CS induced the bridging flocculation
between the emulsion droplets at pH 5.5, which improved the viscoe-
lastic properties of the emulsions. On the other hand, the electrostatic
attraction between OVA and CS increased the dynamic dilatational
elastic modulus (Ed) of absorbed layer at the oil-in-water interface.
These two key points may be used to explain the very stable emulsion at
a lower total biopolymer concentration (0.3%, w/w).
4. Conclusion
This work provided a good understanding for the emulsion stability
of OVA/CS mixtures at pH 4.0 and pH 5.5, especially the contribution
of the rheological properties of the emulsion and the viscoelastic of the
interface adsorbed layer to the emulsion stability. The complexation of
OVA and CS at pH 5.5 improved viscoelastic properties of emulsions,
retarded the mobility of the emulsion droplets, and significantly en-
hanced emulsion stability at a lower total polymer concentration (0.3%,
w/w) even though extensive aggregation of emulsion droplets still oc-
curred. On the other hand, interface rheology results showed that the
addition of CS significantly decreased the diffusion, permeation, re-
arrangement rates of OVA and the interface pressure after adsorption
for 180 min, especially at pH 5.5. OVA/CS complex were able to form
thick adsorption layers around oil droplets at pH 5.5, therefore dilata-
tional modulus of the interfacial layer could be enhanced due to their
interactions. As a result, the emulsion stabilized by the OVA/CS com-
plex at pH 5.5 showed excellent stability. Findings from the present
study may be applied to improve the quality of relevant emulsion
products or fabricate delivery systems for bioactive compounds.
Acknowledgments
The authors acknowledge the financial support from the National
Key R&D Program of China (Program No. 2017YFD0400200), the
Natural Science Foundation of China (NSFC, Grant No. 31772015) and
Wuhan Yellow Crane Special Talents Program.
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