Emulsion Stability and Dilatational Viscoelasticity of
Emulsion Stability and Dilatational Viscoelasticity of
Emulsion Stability and Dilatational Viscoelasticity of
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
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.
⁎
Corresponding author at: College of Food Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China.
E-mail address: [email protected] (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. Food Chemistry 252 (2018) 181–188
ovalbumin and sulphated polysaccharide, leading to a lower surface emulsions were further processed with two passes through a high-
activity (Galazka, Dickinson, & Ledward, 2000). The opposite results pressure homogenizer (AH100B, ATS Engineering Inc. Canada) at
may be ascribed to various physiochemical properties of proteins and 30 MPa. These freshly prepared emulsions were directly subjected to
polysaccharides. analysis or storage stability experiments at 25 °C.
Therefore, for the protein/polysaccharide mixtures, research on the
adsorption behaviour of proteins and the interfacial rheology properties 2.4. Particle size measurement of emulsions
of adsorbed layers can provide a useful insight into the mechanistic
origin of some emulsion properties (Jourdain, Schmitt, Leser, Murray, & The volume-average droplet size distribution (d4,3) of freshly pre-
Dickinson, 2009). Ovalbumin (OVA) is the main protein in egg white, pared emulsions at pH 4.0 and 5.5 were measured by using Malvern
and is commonly used as emulsifier in food industry. Previous studies MasterSizer 2000 (Malvern Instruments Ltd., Malvern, Worcestershire,
showed that the hydrophobicity and net charge of OVA had important U.K.). Before the test, all the emulsions were diluted 100 times using
effects on its interfacial adsorption properties (Croguennec, Renault, phosphate buffer solutions (PBS, 5 mM) with corresponding pH values,
Beaufils, Dubois, & Pezennec, 2007; Delahaije, Wierenga, van the pH values of PBS were adjusted to 4.0 and 5.5 using 0.5 M HCl and
Nieuwenhuijzen, Giuseppin, & Gruppen, 2013; Wierenga, Meinders, 0.5 M NaOH. The refractive index of the emulsions, dispersed phase and
Egmond, Voragen, & de Jongh, 2003, 2005), and OVA/anion poly- continuous phase was set to 1.095, 1.456 and 1.333, respectively. All
saccharide complex coacervates showed a better emulsifying ability measurements were conducted at least in duplicate.
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 2.5. Optical microscopy
and complexes at pH4.0 and pH5.5, respectively (Xiong et al., 2016).
However, the effect of the electrostatic interaction between OVA and CS The morphology of emulsions was studied using Ningbo shunyu CX
on the adsorption behaviour of protein at oil-in-water interface and the 40 optical microscope (China) and the images were captured with a
viscoelastic properties of absorbed layer, and the relationship between OD1400Y digital camera (China).The emulsions were diluted 100 times
this effect and emulsion stability has not been reported. The aim of this using PBS solutions (5 mM) with corresponding pH values, and then
work was to explore the effect of OVA-CS interaction on the emulsion placed on a glass slide with a cover glass for microscopic observation at
stability, and reveal the stabilized mechanism of emulsions prepared by 25 °C.
OVA-CS mixtures at different pH values in the bulk phase. Furthermore,
the interfacial dilatational properties of OVA/CS absorbed layers at oil- 2.6. Creaming index of emulsions
in-water interface were also assessed by the interface rheological
measurements. The creaming stability of emulsions with different formulations at
various pH values was evaluated visually upon quiescent storage for up
2. Materials and methods 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
2.1. Materials height of the whole fresh emulsion (Ht, cm) were recorded at various
periods of storage. The creaming index was calculated using Eq. (1):
Ovalbumin (OVA, A5503, > 98% pure by agarose electrophoresis)
Creaming index (CI, %) = (Hs /Ht) × 100 (1)
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 2.7. Rheological measurement of emulsions
Yunzhou Biochemistry Co., Ltd. (Shandong, China). Soybean oil was
purchased from the local supermarket. Other reagents were obtained The dynamic rheological properties and steady shear viscosities (η)
from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All of emulsions were performed using a strain-controlled rheometer
reagents were analytical grade unless otherwise stated. (AR2000ex, TA Instruments, USA) fitted with parallel plate geometries
(40 mm in diameter). Fresh emulsions were loaded and maintained on
2.2. Preparation of OVA/ CS aqueous complexes 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
OVA (1%, w/v) stock solution was obtained by dissolving OVA loss modulus (G″) of emulsions were measured with varying the fre-
powder in deionized water under gentle stirring at 25 °C for 2 h and quency from 0.1 to 100 rad/s. Strain sweep tests were conducted to
then stood overnight at 4 °C to ensure a complete protein hydration. determine the proper conditions of rheological measurements, and the
Moreover, sodium azide (0.02%, w/v) was added before stirring to strain of all samples was set to 0.5%. The temperature of the rheological
inhibit bacteria growth. CS (1%, w/v) stock solution was prepared by experiment was set at 25 °C. Meanwhile, the viscosity of the emulsions
dissolving CS powder in acetic acid solution (1%, w/v) and stirring at was recorded as the shear rate shifted from 0.1 to 100 1/s.
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 2.8. Dynamic interfacial surface pressure (π) and interface viscoelasticity
then the pH values of OVA alone solution and OVA/CS mixed solution measurement
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/ 2.8.1. Measurement of interfacial surface pressure
CS mixtures and individual OVA. The interfacial surface pressure (π) and adsorption kinetics of OVA
alone or OVA/CS mixtures at the oil/water interface were performed
2.3. Emulsions preparation using an automated drop tensiometer (Tracker-H, Teclis, France) at
25 °C. For this test, to eliminate the interference of impurities in com-
Primary oil-in-water emulsions (100 ml) were prepared by mixing mercial soybean oil, and thus to obtain a clearer description of ad-
the soybean oil (20 ml) and solution of OVA alone or OVA/CS mixtures sorption and diffusion behaviour of the protein molecules at the oil-in-
(80 ml) using a high-speed homogenizer (IKA-ULTRA-TURRAX T25 water interface, medium chain fatty acid (MCT) was used instead of
Wilmington, NC) operating at 10,000 rpm for 1 min. The oil fraction commercial soybean oil for the study of interfacial rheological prop-
was fixed at 20% (w/w). For all emulsions, the total biopolymer was erties. The bulk phase (OVA or OVA/CS mixtures) and oil phase were
fixed at 0.3% (w/w) without salt ion. Furthermore, the primary placed in the cuvette and syringe, respectively. The total biopolymer
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W. Xiong et al. Food Chemistry 252 (2018) 181–188
concentration was fixed at 0.3% (w/w), and the test model was raising emulsions, we assessed the dynamic viscoelastic properties and flow
hanging drop. Before test, dispersions and oil were allowed to stand for behaviour of the fresh emulsions. Generally, the storage modulus re-
at least 1 h to reach 25 °C. The temperature of the system was main- lates to the elasticity of emulsion, which reflects solid properties, and
tained constant by circulating water from a thermostat. The oil droplet loss modulus relates to viscidity of the emulsion, which represents li-
volume was 10 µl during the test. The oil/water interface pressure (π, quid properties. As presented in Fig. 2A and B, the storage modulus (G′)
mN/m) was calculated with Eq. (2): 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),
π (mN/m) = γ0−γ (2)
suggesting the formation of predominantly elastic gel-like emulsions.
where γ0 (mN/m) and γ (mN/m) is the interfacial tension of pure oil- Specially, the G′ value of emulsion stabilized by OVA/CS complex at pH
distill water (26 mN/m) and OVA alone or OVA/CS mixed solutions, 5.5 was nearly 10 times greater than the other emulsions, and the G′
respectively. The surface tension was determined by drop shape ana- was almost independent on the sweep frequency in the test range of
lysis (Benjamins, Cagna, & Lucassen-Reynders, 1996). All measure- 0.1–10 rad/s. Additionally, it was worthy to note that the G′ of emul-
ments were performed up to 3 h. 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
2.8.2. Measurement of interfacial viscoelasticity pH5.5 than pH 4.0 (Benichou, Aserin, & Garti, 2002; Dickinson, 1998;
To obtain surface dilatational parameters, the dynamic interface Dickinson & Pawlowsky, 1997). These results were consistent with the
viscoelasticity of OVA alone and OVA/CS mixtures at the oil–water creaming stability of emulsion, and suggesting that higher G′ could
interface were investigated using an automated drop tensiometer provide great creaming stability due to the droplet flocculation.
(Tracker-H, Teclis, France) at 25 °C. OVA alone or OVA/CS mixtures In addition, apparent viscosities of fresh emulsions as a function of
and oil were placed in the cuvette and syringe, respectively. The total shear rate (0.1–100 1/s) in the absence or presence of CS at pH 4.0 and
biopolymer concentration was fixed at 0.3% (w/w). The sinusoidal pH 5.5 were illustrated in Fig. 2 C. As expected, all fresh emulsions
interfacial compression and expansion were performed by changing the showed pseudo plastic flow behaviour in the whole shear rate range,
drop volume at 10% of deformation amplitude within the linear regime, indicating deflocculation of associated droplets in the emulsions (Liu &
and the oscillation frequency was 0.1 Hz, the droplets volume was Tang, 2014). On the other hand, with the increase of shear rate, the
10 µl, and the experiment was started after equilibration 60 s. The emulsion droplets and polymer chains became more ordered along the
number of active and blanks was 5 cycles during the experiments. flowing direction, which reduced the resistance to flow observed as a
Details of this experiment were described by Perez, Carrara, Sánchez, lower viscosity. However, significant differences were still existed
Santiago, and Patino (2009). among the emulsions with different formulations. Adding CS resulted in
a higher viscosity of emulsions compared with that stabilized by pure
2.9. Statistical analysis 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
Unless otherwise stated, all measurements were carried out in tri- emulsion droplets. Specially, the emulsion stabilized by the OVA/CS
plicate. One way analysis of variance (ANOVA) with a 95% confidence complex at pH 5.5 had the highest apparent viscosity, especially at
interval was used to compare the significance of the results obtained. relatively low shear rate of 0.1–3 1/s, which greatly retarded the mo-
Statistical analysis was performed using SPSS software version 19.0. bility of the emulsion droplets and thus inhibited the creaming of
emulsions. These results were consistent with the emulsions stabilized
3. Results and discussion by sodium caseinate/flaxseed gum mixtures (Zhao et al., 2015). These
findings indicated that the flocculation could weaken the flow of
3.1. Microstructure and oil droplets size of emulsions emulsions by resistance force among interparticles, thereby increasing
the apparent viscosity of emulsions.
The microstructure, average oil droplets size distribution (d4, 3) of
fresh emulsions stabilized by OVA alone and OVA/CS mixtures are 3.3. Creaming stability
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 Fig. 3 showed the effect of CS addition on the creaming stability of
much closer to pI of OVA than pH 4.0. Therefore, the electrostatic re- emulsions at pH 4.0 and pH 5.5. There was obvious difference in the
pulsion between the droplets was insufficient to prevent flocculation at plots between the OVA stabilized emulsions with CS at pH 5.5 and the
pH 5.5 (Fig. 1B). Additionally, compared to the OVA stabilized emul- OVA stabilized emulsions with or without CS at pH 4.0. In the absence
sions, the incorporation of CS caused a wider size distribution of of CS, the creaming layer was soon observed, which might be attribute
emulsion droplets, and resulted in the increased droplets size from 21.2 to the low net charge of OVA (data not show). However, the creaming
to 33.8 µm (d4, 3) at pH 5.5 (Fig. 1D). index (CI, %) of OVA stabilized emulsion at pH 4.0 was higher than that
However, optical microscopy results showed extensive flocculation at pH 5.5. This result might be due to the fact that pH 5.5 was closer to
of emulsion droplets in the presence of CS at pH 5.5. Indeed, OVA was the isoelectric point of OVA, and the aggregation degree of protein
negatively charged while CS was positively charged at pH5.5, and ul- molecules was higher than pH 4.0, resulting in greater steric hindrance
timately forming the complexes with positive charge by electrostatic and improving the stability of the emulsion. What’s more, significant
attraction (Xiong et al., 2016). On the other hand, our past work sug- creaming behaviour was also observed at pH 4.0 for the emulsion in the
gested that the optimum mass ratio of OVA/CS was 3.6:1 at pH 5.5 by presence of CS, and the CI value was slightly greater than the emulsion
the isothermal titration calorimetry (ITC) (Xiong et al., 2016). This without CS at pH 5.5. Specifically, in the case of adding CS at pH 5.5, it
indicated that the surface of emulsion droplets was not completely was surprisingly observed that no creaming occurred during the whole
coated with CS, which led to the occurrence of bridging flocculation storage up to 14 days, indicating extraordinary stability against
(Dickinson, 2008). At pH4.0, both OVA and CS were positively charged, creaming, although the flocculation of emulsion was observed from the
and no aggregation were observed due to the sufficient electrostatic microstructure and droplet size distribution as shown in Fig. 1D. In-
repulsion and steric hindrance. deed, the addition of CS increased the viscosity of the bulk phase and
effectively hindered the free movement of the emulsion droplets
3.2. Rheological properties of emulsions (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
To further investigate the macroscopic characteristics of the viscoelastic property at pH 5.5 (Fig. 2). This correlates well with the
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W. Xiong et al. Food Chemistry 252 (2018) 181–188
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 diffusion, actual adsorption (penetration and unfolding) and con-
flocculation. When polysaccharide concentrations well below satura- formational reorganization (Dickinson, 2011). Generally, there is a
tion coverage, the formation of polymer-cross-linked droplets network rate-determining step, showing relatively low interfacial surface pres-
induced by bridging flocculation makes the emulsions have a gel-like sures during the first step (Camino, Pérez, Sanchez, Patino, & Pilosof,
rheological property, thus exhibiting excellent stability (Cao, 2009). The change in interfacial surface pressure (π) with adsorption
Dickinson, & Wedlock, 1990). time (t) can be correlated by a modified form of Wardand Tordai
Moreover, the reversed emulsions after storage for 14 days still had equation (Ward & Tordai, 1946):
a great stability as observed from the inserted digital photos in Fig. 3.
This finding suggested that a gel-like network structure of emulsions π = 2C0 KB T (D t/3.14)1/2 (3)
was formed involving extensive droplet flocculation, and thereby pro- where C0 is the concentration in the continuous phase, KB is the
viding extraordinary creaming stability for the emulsion stabilized by Boltzmann constant, T is the absolute temperature, and D is the diffu-
OVA/CS complex at pH 5.5. This phenomenon was also observed in the sion coefficient. If the adsorption process is controlled by the protein
emulsions stabilized by preheated soy protein (Liu & Tang, 2014; Tang diffusion, a plot of π versus t1/2 will then be linear, and the slope of this
& Liu, 2013) or whey protein (Liu & Tang, 2011), and chitin Nano plot will be the diffusion rate (Kdiff) (MacRitchie, 1978), as shown in
crystal particles (Tzoumaki, Moschakis, Kiosseoglou, & Biliaderis, Fig. 4A.
2011). Furthermore, the first-order equation can be used to analyze the
rates of penetration and rearrangement of adsorbed protein molecules
3.4. Interfacial adsorption and dilatational rheological properties at the interface (Graham & Phillips, 1979):
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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
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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).
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.
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.
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interfacial absorbed layer, especially at pH 5.5, even though OVA triacylglycerol/water interfaces covered by proteins. Colloids and Surfaces A:
concentration was lower than the pure OVA systems. This phenomenon Physicochemical and Engineering Aspects, 114, 245–254.
Camino, N. A., Pérez, O. E., Sanchez, C. C., Patino, J. M. R., & Pilosof, A. M. (2009).
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and polysaccharide could improve the mechanical strength of oil – namics at the air–water and oil–water interfaces. Food Hydrocolloids, 23(8),
water interfacial absorbed layer. This interfacial property was posi- 2359–2368.
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The authors acknowledge the financial support from the National Interfacial dynamic properties of whey protein concentrate/polysaccharide mixtures
at neutral pH. Food Hydrocolloids, 23(5), 1253–1262.
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