Food Hydrocolloids 111 (2021) 106211

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

Effect of pH, ionic strength, chitosan deacetylation on the stability and

rheological properties of O/W emulsions formulated with chitosan/
casein complexes
Fang Zhang 1, Xixi Cai 1, Lan Ding , Shaoyun Wang *
College of Biological Science and Technology, Fuzhou University, Fuzhou, 350108, China

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

Keywords: Emulsions stabilized by polysaccharide/protein complexes have attracted great interest. However, their emulsion
Chitosan properties and interfacial viscoelastic behavior need to be further investigated. Here, emulsion stabilized by
Casein chitosan/casein complexes was used as a model system and the effects of chitosan concentration, pH, ionic
Complexes
strength, and chitosan deacetylation degree on the rheological properties and stability of emulsion were studied.
Emulsion
Stability
The presence of chitosan could effectively improve the emulsification activity of casein and the stability of the
Rheology obtained emulsion. The emulsion stabilized by chitosan/casein complexes showed broad emulsification pH range
from 3.5 to 6.5. The rheological properties of the chitosan/casein complexes were proved to be primarily affected
by chitosan, showing great shear resistance, fast adsorption interface capability, and predominantly viscous
characteristics. The loss modulus (G′′ ) was much larger than the storage modulus (G′ ) in all the rheological tests,
suggesting that the stability of the complexes stabilized emulsion mostly depended on the viscosity effects, rapid
adsorption characteristics and spatial effects of biopolymer structures. These findings might help developing
stable, controllable polysaccharide/protein emulsification systems.

1. Introduction combined properties of protein and polysaccharide (Dickinson, 2009;

Lv, Fu, Zhang, & Wang, 2019). The protein-polysaccharide complexes
Protein and polysaccharide as natural biopolymers have been exhibit substantially enhanced emulsifying properties compared to the
applied in various fields such as food, cosmetics, and pharmaceuticals protein (O’Regan & Mulvihill, 2010; Wooster & Augustin, 2006).
(Li, Fang, Al-Assaf, Phillips, & Jiang, 2012). Proteins are widely used as Different combinations of polysaccharide/protein are possible to display
emulsifiers due to their excellent surface activities and amphipathic unique emulsification effect depending on their specific structure and
properties. However, an inherited drawback for them is the low solu­ characteristics. However, the stability mechanism of poly­
bility at pH values close to their isoelectric points (pI), leading to a saccharide/protein complexes is complicated because of the
reduced pH range where they exhibit satisfactory emulsifying activity. protein-polysaccharide interactions, especially at the interface, which
And the resulted poor resistance towards environmental stress (pH would affect the adsorbed layer by changing viscoelastic properties.
changes, ionic strength, and so on) extremely limited their practical Thus, it is crucial to study the emulsion stability mechanism and inter­
applications (Evans, Ratcliffe, & Williams, 2013; Lam & Nickerson, face interactions of polysaccharide/protein complexes.
2013). Different from proteins, which can adsorb at the oil-water Casein (CA) is the major component of milk proteins and commonly
interface, polysaccharides usually have weak surface activities as their used in the food industry for their excellent emulsifying and other
strong hydrophilic character, but they can also behave as emulsion functional properties. The excellent emulsifying properties of casein
stabilizers by forming an extended network and thus affecting the mainly depend on its high hydrophobicity and excellent surface activity
continuous phase viscosity (Cai et al., 2018). Therefore, one emerging (Horne, 2002). Caseins can adsorb onto the interface quickly and sta­
interest in developing polysaccharide/protein complexes as novel bilize the emulsion through electrostatic and steric repulsion. However,
emulsifiers and stabilizers has been raised, to make full use of the at the pH value around its average pI 4.5, their net surface charge is zero,

* Corresponding author.
E-mail address: [email protected] (S. Wang).
1
These authors contribute equally to the article.

https://doi.org/10.1016/j.foodhyd.2020.106211
Received 23 December 2019; Received in revised form 20 July 2020; Accepted 24 July 2020
Available online 29 July 2020
0268-005X/© 2020 Elsevier Ltd. All rights reserved.
F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

and caseins’ emulsifying ability is lost, leading to flocculation. To avoid study, we’d like to further investigate the properties of emulsion stabi­
this, attention has been paid to employ the complexes of casein and lized by chitosan/casein complexes and evaluate the effects of chitosan
polysaccharide as an emulsifier, taking advantage of the adsorbed concentration, pH, ionic strength and chitosan deacetylation degree.
crosslinking layer might be formed to enhance the o/w emulsion sta­ Moreover, we will illustrate the stability mechanism from the rheolog­
bility (Li, Fang, Phillips, & Al-Assaf, 2013; Xu, Wang, & Ping, 2017). For ical behavior of chitosan/casein at oil/water interface.
example, the casein/xanthan gum complexes at the oil-water interface
could stabilize the emulsion over a wider range of pH and exhibit better 2. Materials and methods
ionic strength resistance than casein or xanthan gum alone (Liu et al.,
2012). 2.1. Materials
Chitosan (CS), a natural cationic polysaccharide, is one of the most
abundant polysaccharides in the world and is commonly obtained by Casein (BR) was provided by Shanghai Macklin Biochemical Co., Ltd
deacetylation of chitin (Hu, Wang, Li, Zeng, & Huang, 2011; Santos (Shanghai, China). Chitosan (low molecular weight) was purchased
et al., 2018). It exhibits pH-responsive behavior due to the large from Sigma-Aldrich Life Science & Technology Co., Ltd (Wuxi, China).
amounts of amino groups on its chain. Its pKa is affected by the degree of Medium-chain triglycerides, Nile blue, and Nile red were obtained from
deacetylation and generally locates at about 6.5 (Yuan, Jacquier, & Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). All the
O’Riordan, 2018). Its amphiphilic polyelectrolyte nature makes it ideal other chemicals used were of analytical grade and commercially avail­
for stabilizing emulsions, combing both electrosteric and viscosifying able. Deionized water was used throughout to prepare all solutions.
stabilization mechanisms (Asfour, Elmotasem, Mostafa, & Salama, 2017;
Reis, Yoshida, Reis, & Franco, 2011; Rodrı ́guez, Albertengo, & Agulló, 2.2. Preparation of oil-in-water (O/W) emulsion
2002). On one hand, it can stabilize emulsions by adsorption at the oil
droplet surface through electrostatic and/or steric repulsive forces, 2.2.1. Chitosan/casein complexes
preventing droplet flocculation and coalescence. On the other hand, it Chitosan and casein stock solutions were prepared as described in
can improve emulsion stability by changing the viscosity of the aqueous our previous report (Ding et al., 2019), and diluted to different con­
continuous phase, slowing down droplet movement and encounters. centrations (0.2, 0.6, 1.0, 2.0, 3.0, 4.0, 5.0 and 6.0 mg/mL) as needed.
Thanks to its large molecular weight and linear unbranched structure, it Chitosan/casein complexes with different mass ratios were prepared by
can be used as an effective viscosity enhancer, even in acidic environ­ adding chitosan solutions into casein solutions of different concentra­
ments. It was found that chitosan could reduce the interfacial tension by tions at 1:1 vol ratio at pH 5.5 and kept being stirred for 1 h at 500 rpm.
adsorbing onto the interface of air-water/oil-water, and slow oil droplet Chitosan with different deacetylation degree was prepared according to
diffusion by forming a dense polyelectrolyte network in the continuous the previous work (Huang, Cai, & Lapitsky, 2015).
phase (Bouyer, Mekhloufi, Rosilio, Grossiord, & Agnely, 2012; Zhang,
Zhou, & Cheng, 2015). 2.2.2. Emulsion stabilized by chitosan/casein complexes
Conventionally, the fabrication of polysaccharides/protein com­ To get the visualization of the interface surrounding oil droplets, the
plexes is mainly based on the attractive electrostatic interactions be­ emulsions were prepared according to Surh et al. (Surh, Decker, &
tween anionic polysaccharides and proteins bearing positive charge McClements, 2006) with some modification. The dispersions (95 wt%)
below their pI (Zhang, Zhang, Fang, & Wang, 2019). Given the good and MCT (5 wt%) were mixed using a high-shear mixer (FJ200-SH,
availability, polycationic chitosan has gained more and more attention Specimen Model Factory, Shanghai) for 2 min at 10,000 rpm to get
and been employed as one of the composites in the biopolymer emulsion pre-emulsion. Then pre-emulsion was passed through a high-pressure
stabilization system in recent years. Laplante, Turgeon, and Paquin homogenizer, cycled three times at 70 MPa (AH-2010, ATS, Canada).
(2006), for example, studied the emulsion-stabilizing properties of chi­ Sample for salt stability study (at pH 5.5) was prepared with different
tosan in the presence of whey protein isolate as a function of mixture amounts of NaCl (0, 50, 100, 150, 300 mM). As for pH stability study,
ratio, ionic strength, and pH. Yuan et al. (Yuan, Gao, Decker, & samples were adjusted to the desired pH value by using either HCl (0.1
McClements, 2013) explored the effect of chitosan addition on the or 1 M) or NaOH (0.1 or 1 M).
physicochemical properties of whey protein coated lipid droplets. The
properties of emulsions stabilized by sodium caseinate-chitosan com­ 2.3. Characteristics and stability of emulsions stabilized by chitosan/
plexes have also been preliminarily discussed (Zinoviadou, Scholten, casein complexes
Moschakis, & Biliaderis, 2012). However, the effect of chitosan deace­
tylation degree (DD) on the emulsifying properties of chitosan/protein 2.3.1. Dynamic light scattering (DLS)
complexes has seldom been discussed. DD of chitosan has been reported The particle size, polydispersity index (PDI) of all emulsions were
to affect the solubility, degradation rate as well as the encapsulation determined by DLS at a backscatter detection angle of 173◦ to avoid
efficiency when chitosan was employed as gene carrier (Bozkir & Saka, multiple light scattering. The zeta-potential was measured by electro­
2004). It has also been proved to have a significant relationship with the phoretic light scattering (ELS) using the Smoluchowski model. The
emulsification properties (Del Blanco, Rodriguez, Schulz, & Agulló, emulsion samples were diluted 1:100 in deionized water with the same
1999). With the increase of DD, both of the charge density along the salt concentration and pH as the droplets originally dispersed. All
chitosan chain and its hydrophobicity increased. We believe the study of measurements were performed with a Malvern Zetasizer after 1 h since
DD effect would be important for the stability of chitosan involved emulsion prepared (Nano ZS, Malvern Instruments Ltd., Worcestershire,
polysaccharide/protein emulsion system. Besides, a comprehensive UK).
study on the emulsifying properties of chitosan/casein complexes, and
especially, the interaction of chitosan/casein at the oil/water interface 2.3.2. Creaming stability
still needs to be further investigated. Studies on its systematic emulsi­ Ten milliliters of each emulsion sample was transferred into a cy­
fying properties and emulsification stabilization mechanism would lindrical test tube (internal diameter 1 cm, height 10 cm) and then
provide fundamental information for popularizing chitosan/casein stored at room temperature (25 ◦ C) for one month. During storage,
complexes as an emulsifier in practical use and inspired ideas for even emulsions usually separated into a thin “creamed” layer at the top and a
other protein/polysaccharide system. In our previous report, we have transparent “serum” layer at the bottom over time. The creaming sta­
established a phase diagram and studied the complexation behavior of bility of the emulsion samples was determined by visual observation of
casein and chitosan in different conditions, including pH, ionic strength the total height of the emulsion in the tubes (HE) and the height of the
and chitosan charge density (Ding, Huang, Cai, & Wang, 2019). In this serum layer (HS) formed at the bottom of the test tube (Mwangi, Ho,

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F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Fig. 1. Effect of chitosan concentration on the (A) size and PDI, (B) Zeta-potential and CI of emulsion stabilized by chitosan/casein complexes at 1 mg/mL casein.
The blue shaded area represents flocculation. (C) The photography of chitosan/casein complexes stabilized emulsion at 1 mg/mL casein and different chitosan
concentrations. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Tey, & Chan, 2016). The creaming index (CI) was calculated as follows under the same condition. The final interfacial rheological data were
(Eq. (1)): analyzed using Rheoplus software version 3.21 (Anton-Paar).
The rheology experiments were conducted at least thrice, and the
HS
CI(%) = × 100% ​ (1) others were all conducted in triplicate. The data represented as the
HE
mean ± standard deviation where feasible.
Sodium azide (0.02% final concentration) was added into emulsions
to prevent microbial growth (Chang, Gupta, Timilsena, & Adhikari, 3. Results and discussion
2016). And the emulsion photographs were taken after one-month
storage at ambient temperature. 3.1. Effect of chitosan concentration on emulsion stabilized by chitosan/
casein complexes

2.4. Interfacial rheology measurements To investigate the effect of chitosan concentration on the colloid
stability as well as creaming stability of emulsion stabilized by chitosan/
The interfacial rheology of chitosan/casein complexes at the oil/ casein complexes, 2 mg/mL casein solution was mixed with chitosan at
water interfaces was measured using a rheometer (Anton-Paar MCR 302, different concentrations (0–6 mg/mL) to obtain emulsions with different
Graz, Austria) equipped with Bi-cone interfacial rheology system at 25 chitosan/casein mass ratios. As shown in Fig. 1A, when the concentra­
◦
C. The temperature was controlled with a Peltier system. The double tion of chitosan <0.3 mg/mL, flocculation formed. As the increase of
cone used had a radius of 34.14 mm, and the angle of the cone was 5◦ . chitosan concentration from 0.3 to 3 mg/mL, the emulsion diameter
About 118 mL of lower phase chitosan/casein complexes solution was increased from 543 nm to 812 nm. That’s because, in the presence of a
placed in an interfacial rheological cell (80 mm in diameter), and the low concentration of chitosan, few of them form the complexes, making
ring was lowered to contact with the lower phase surface. To increase most of the droplet surface was absorbed with casein. The chitosan
repeatability, zero-gap calibration was conducted before the lower presented in the continuous phase can easily cause bridging floccula­
phase added, and the gap was kept constant. Subsequently, upper phase tion, which would be dominant when the chitosan concentration is low
MCT was carefully poured on the top of the lower phase. The air bubbles and destabilize the emulsion. Similar results have also been reported in
should be avoided during sample loading. The inertia determination and polysaccharides like pectin, carrageenan, or chitosan involved protein
MSC (Micro Stress Control) calibration was performed before starting stabilized emulsions (Guzey & McClements, 2007; Hong & McClements,
each series of measurements to optimize the measurement results. 2007). The bridging effect went weaker as the concentration of casein
Dynamic time sweeps were performed with γ (strain amplitude) = increased to 0.5 mg/mL, where the complexes were saturated with
1% (in the linear region) and ω (angular frequency) = 1 rad/s for 8 h. chitosan. As demonstrated in our previous work (Ding et al., 2019), the
Dynamic strain sweeps were conducted with γ = 0.1–100%, ω = 1 rad/s. change of complexes diameter has a positive correlation with the chi­
Frequency-sweep oscillatory shear tests were carried out with γ = 1% (in tosan concentration. Consequently, a larger complex size led to larger
the linear region), ω = 0.1–20 rad/s. Flow curves of steady-shear vis­ stabilized droplets. The emulsion PDI was less affected by the chitosan
cosity were obtained at γ̇(shear rates) = 0.1–100 s− 1. concentration and all maintained at around 0.3, showing good dis­
To get interfacial rheological data such as storage modulus (G′ ) and persibility. This was consistent with the microstructure results obtained
loss modulus (G′′ ), all tests used Bi-cone interfacial rheology system with confocal laser scanning microscopy (Fig. S1), showing emulsions
should be replicated with the concentric cylinder measurement system

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F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Fig. 2. Effect of pH on the (A) size; (B) PDI; (C) zeta-potential and (D) CI of emulsion stabilized by chitosan/casein complexes. The blue shaded area represents
flocculation; (E) Photography of casein stabilized emulsion; (F) Photography of chitosan/casein complexes stabilized emulsion. (For interpretation of the references
to colour in this figure legend, the reader is referred to the Web version of this article.)

stabilized by casein and chitosan/casein complexes were all in a stability of an emulsion. The higher the emulsion stability it is, the lower
spherical shape and distributed uniformly. the creaming index would be. As shown in Fig. 1B, with the increase of
With the increase of chitosan concentration from 0.3 to 3 mg/mL, the chitosan concentration from 0.3 mg/mL to 3 mg/mL, the creaming index
zeta-potential of emulsion raised from +31.4 mV to a constant value at of the emulsion decreased from 85.6% to 5.8%, indicating that the
+45 mV as the polysaccharide concentration exceeded 0.5 mg/mL (CA: addition of chitosan improved the stability of the emulsion effectively.
CS = 1:0.5) (Fig. 1B). This trend was similar to our previous studies Chitosan was considered to provide stronger electrostatic repulsion and
about chitosan/casein complexes, where the casein micelle surface was steric hindrance between the emulsion particles, making the emulsion in
gradually covered by chitosan with a zeta-potential increase and a bal­ a stable equilibrium state. Besides, chitosan has a certain viscosity,
ance crossover point at certain chitosan concentration, revealing the full which can change the properties of the continuous phase and help to
cover of casein surface by chitosan at this mass ratio (Ding et al., 2019). slow down the droplet’s movement and encounters, thereby improving
However, different from the chitosan/casein complexes, the surface the stability of the emulsion. It was also clearly observed from the
potential of which reached a stable value at 0.1 mg/mL chitosan, more emulsion photograph (Fig. 1C) that the stability of the emulsion was
chitosan (>0.5 mg/mL) was needed to reach the saturation point in the positively correlated with the chitosan concentration.
case of emulsion stabilized by chitosan/casein complexes. This can be
attributed to the increased particle size of casein coated around the oil 3.2. Effect of pH on emulsion stabilized by chitosan/casein complexes
droplet that needs more chitosan to cover the casein surface fully. It is
noteworthy that the emulsion flocculated and precipitated when the As the electrostatic interaction between protein and polysaccharide
chitosan concentration used was 0.1 mg/mL. This might be due to the is highly dependent on pH, it is essential to investigate the effect of pH
bridging flocculation of chitosan/casein complexes close to this on the colloid stability and creaming stability of chitosan/casein stabi­
concentration. lized emulsion as well. As shown in Fig. 2A, the particle sizes of emulsion
Creaming index is one of the main parameters related to the long stabilized by casein were kept around 300 nm at pH range from 2.5 to

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F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Fig. 3. Effect of NaCl concentration on the (A) size and PDI; (B) zeta-potential and CI of emulsion stabilized by chitosan/casein complexes at 1 mg/mL casein and the
mass ratio of casein: chitosan was 1:1.5.

6.5, except the situation around casein’s PI. By contrast, the particle size and F). We reasoned the pH effect towards emulsion stability for the
of emulsion stabilized by chitosan/casein complexes first decreased electrostatic interaction change. As discussed above, when the pH is
from 946 to 621 nm at pH 2.5 to 4.5 and then increased to 938 nm at pH lower than the pI of casein, chitosan would more likely be free in the
6.5. It’s reasonable because at the pH < 4.5, both casein and chitosan continuous phase working as only a thickening agent. With a proper
carry positive charges and there would be electrostatic repulsion be­ concentration such presence of chitosan be helpful for the stability of
tween them. Casein would be prone to adsorb on the surface of the emulsions. However, when the concentration of chitosan is high enough,
droplets playing the role of emulsifier. While the chitosan is likely to and the thermodynamic incompatibility effect went stronger, floccula­
work as a thickening agent. Non-adsorbed chitosan in the continuous tion happened resulting in bigger particles in size, which according to
phase induced depletion flocculation leading to larger droplets in size. the Stokes’ law would significantly promote creaming velocity. As the
As the increase of pH, the repulsion progressively became an attraction increase of pH, electrostatic interaction between the surface-active
and droplet surface was stabilized by the non-covalent complex of chi­ casein and chitosan became stronger and the steric hindrance as well
tosan/casein. The electrostatic interaction became stronger and the as electrostatic repulsion gradually dominant making the emulsion
complexes went more compact at pH 4.5, where the size of droplets was stability improved. Bouyer et al. (Bouyer et al., 2012) for example, re­
smallest. These results were similar to the previous report about konjac ported that the stronger electrostatic interaction could grant chito­
glycoside (Lu, Zheng, & Miao, 2018). Remarkably, in contrast to the san/whey protein isolate emulsion with longer stability.
chitosan-free emulsion, no obvious creaming was observed in chito­
san/casein complexes stabilized ones at the pI of casein between 4.5 and 3.3. Effect of ionic strength on emulsion stabilized by chitosan/casein
5.0. The effect of pH on the emulsion PDI was consistent with the trends complexes
observed in size-changing (Fig. 2B). It is similar to the results obtained in
chitosan Pickering emulsion studied by Wei et al. (Wei Z 2012). In a To investigate the effect of ionic strength, the emulsion stability was
word, the pH stability of emulsion was improved, and the pH application monitored at various NaCl concentrations. As shown in Fig. 3A, with the
range was broadened by using chitosan/casein complexes effectively. addition of NaCl (0–300 mM), the particle size of emulsion stabilized by
As shown in Fig. 2C, the zeta-potential of emulsion stabilized by chitosan/casein complexes increased from 768 to 996 nm, while the PDI
casein changed from positive to negative as pH increased from 2.5 to 6.5 value remained around 0.2. The zeta-potential of the emulsion
with a crossover point at pI, while the zeta-potential of emulsion sta­ decreased with the increase of ionic strength (Fig. 3B), which can be
bilized by chitosan/casein complexes decreased from +53.6 to +40.4 attributed to the electrostatic screening effect of NaCl. At high NaCl
mV at such pH range, which can be owing to the deprotonation of chi­ concentration, the salting-out effect was dominated, causing emulsion
tosan. Meanwhile, the stability of the two kinds of emulsions at different flocculated and thus the particle diameter increased. Besides, the
pH was presented in Fig. 2D. Except for flocculation at pI, the casein creaming index increased from 7.93% in salt-free solution to 26.36%
stabilized emulsion at other pHs occurred different degrees of creaming when 300 mM NaCl was present (Fig. 3B).
after one-month storage, suggesting that the casein stabilized emulsion
prepared at present oil-water ratio didn’t possess long-term stability
3.4. Effect of deacetylation degree of chitosan on emulsion stabilized by
(Fig. 2D and E). By contrast, except obvious stratification at pH 2.5, the
chitosan/casein complexes
creaming index of emulsion stabilized by chitosan/casein complexes
remained below 40% and was less than 10% at pH 4.5 and 5.5 (Fig. 2D
To illustrate the effect of the chitosan deacetylation degree on the

Fig. 4. Effect of deacetylation degree of chitosan on the (A) size; (B) PDI; (C) zeta-potential; (D) CI of emulsion stabilized by chitosan/casein complexes at 1 mg/mL
casein and the mass ratio of casein: chitosan was 1:1.5.

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F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Fig. 5. Shear strain dependence of (A) the storage modulus (G′ ) and (B) the loss modulus (G′′ ) of casein, chitosan, chitosan/casein complexes at pH 5.5.

emulsion, the performance of the emulsion at pH 5.5 with chitosan

deacetylation degree between 61.21 and 87.72% were monitored. As
shown in Fig. 4A, the size of the emulsion had no significant difference at
the tested DD range and the PDI dropped slightly. The increased chito­
san DD resulted in an elevated zeta-potential and reached +44.35 mV
when using chitosan with 87.72% DD, which was due to the increased
charge density of chitosan with higher DD. Meanwhile, the creaming
index reduced from 10.80 to 7.92% as the chitosan deacetylation degree
increased from 61.21 to 87.72%. The emulsion with high deacetylated
chitosan had stronger electrostatic repulsion between particles which
could inhibit flocculation. Besides, the high DD chitosan was more
viscous, which would also help prevent particles from coalescence by
slowing down film drainage. The results were in good agreement with
previous research, suggesting the stability of the emulsion stabilized by
chitosan/casein complexes was related to the chitosan deacetylation
degree (Li & Xia, 2011). Fig. 7. The interfacial viscosity curves of casein, chitosan, chitosan/casein
complexes at pH 5.5.

3.5. Rheological properties chitosan, and fairly higher than that of casein, indicating that the
properties of the chitosan/casein complexes were greatly affected by
3.5.1. Amplitude sweep chitosan. However, the viscoelastic modulus (G′ and G′′ ) of the chito­
Shear strain sweeps are typically used to reflect the ability of the san/casein complexes reduced significantly in the presence of NaCl. This
interface to resist shear. In general, when the droplet collides, the could be ascribed to the weaker interactions between the adsorbed two
interface film would rupture, and the emulsion will be unstable. polymers at the interface, which was consistent with the result reported
Therefore, the maximum shear stress applied to the interface without by Tan et al. (Tan et al., 2017).
destroying the structure can be checked by the shear strain sweeps. As
shown in Fig. 5, with the increase of shear strain, the storage modulus 3.5.2. Frequency sweep
(G′ ) and loss modulus (G′′ ) of the chitosan/casein interface had no The frequency sweep of the chitosan/casein complexes was per­
obvious changes, remained at around initial value, suggesting that the formed in a range of angular frequencies from 0.1 to 20 rad/s in a linear
linear viscoelastic region of the samples was very wide and the formed viscoelastic region of 1% (Fig. 6). The frequency results of the absorbed
interface layer possessed good shear resistance. However, it was worth layers formed by different biopolymers were quite similar. With the
noting that the G′ of all the samples tested were much smaller than its increase of angular frequency, the G′ and G′′ of chitosan, casein, and
G′′ , which reflected the interfacial films were majorly affected by the G′′ their complexes all showed a positive proportional increase, and the G′′
of samples. A similar phenomenon was found in the interfacial rheology was much larger than G’. This was consistent with the study of Cho et al.
of corn fiber gum and octenyl succinate anhydride-modified starch, (Cho, Heuzey, Bégin, & Carreau, 2006), regarding the rheological fre­
whose absorbed layers were predominantly viscous (Jin et al., 2017). quency sweep of chitosan. Also, the modulus curve of the
The G′ and G′′ of the chitosan/casein complexes were close to that of

Fig. 6. Frequency sweep of (A) the storage modulus (G′ ) and (B) the loss modulus (G′′ ) of casein, chitosan, chitosan/casein complexes at pH 5.5.

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F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Fig. 8. The time sweep of (A) interfacial storage modulus (G′ ) and (B) interfacial loss modulus (G′′ ) of casein, chitosan, chitosan/casein complexes at the oil/water
interface (pH 5.5).

chitosan/casein complexes was basically coincident with chitosan, properties of chitosan, which could not only reduce surface tension and
while the casein had a higher G′ and G′′ when the angular frequency was form a viscoelastic film, but also change the mobile phase (Calero N
below 10 rad/s. This might be related to the structure of casein and 2010).
chitosan, resulting in a different arrangement at the interface.
4. Conclusion
3.5.3. Viscosity
The apparent interfacial shear viscosities of the absorbed films In this study, the effects of complexes ratio, pH, ionic strength,
formed by four different biopolymers in a range of shear rates (10− 1≤γ deacetylation degree on the properties, and stability of emulsion stabi­
≤ 10 s− 1) were shown in Fig. 7. The interfacial viscosity of all the sys­ lized by chitosan/casein complexes were investigated. Results showed
tems tested showed high viscosities and remarkable shear thinning that the presence of chitosan could effectively improve the emulsifica­
characteristics of pseudoplastic fluids. The shear-thinning observed tion activity of casein and the stability of emulsion obtained. The pH
were very common in the polymer formed films (Li et al., 2016; Lu et al., emulsification range could be effectively broadened by using chitosan/
2018). A relatively high interfacial viscosity was observed in samples casein complexes instead of casein. The instability of emulsion has been
with chitosan or chitosan/casein complexes indicating the stronger significantly improved at casein’s pI, providing a possibility for the
interaction between molecules presented. With high salt concentration application of the emulsion stabilized by chitosan/casein complexes in
(300 mM NaCl), the viscosity at the low shear rate was increased, and we acidic environments. The interfacial rheology studies of chitosan/casein
reasoned this for the salt-induced casein rearrangement on the interfa­ complexes showed that the rheological properties of the complexes were
cial surface. Like previous reports (Sharma, Jaishankar, Wang, & mainly affected by chitosan, having great shear resistance, fast adsorp­
McKinley, 2011), a shear-thinning effect on the liquid-liquid interfacial tion interface capability, and specific viscosity characteristics. The sta­
surface was observed in all these four treatments. However, when the ble mechanism of chitosan/casein complexes towards the emulsion
shear rate exceeded 10 s− 1, no more shear-thinning behavior and the system is mainly dependent on the viscosity effects, rapid adsorption
difference between samples was observed. characteristics, and steric stabilization effects profited from the partic­
ular structures of biopolymer used. The information on chitosan/casein
3.5.4. Isothermic interfacial complexes stabilized emulsion obtained in this work provided support
Generally, the stability of emulsion was influenced by the molecule for future application of such complexes and could potentially benefit
adsorption rate. The time sweep experiment provided a visual insight the design of other protein/polysaccharide complexes stabilized emul­
into the process of molecule adsorption at the oil/water interface. As sion system.
shown in Fig. 8, the interfacial G′ and G′′ were unchanged basically
overtime for all tested systems, which indicated that the samples ach­ CRediT authorship contribution statement
ieved equilibrium immediately after reaching the interface. The shear
elastic modulus was correlated with the structure of tested samples. The Fang Zhang: Conceptualization, Methodology, Writing - review &
polymers that possessed a flexible structure and changed conformation editing. Xixi Cai: Formal analysis, Writing - original draft. Lan Ding:
more easily could complete the interfacial adsorption in a short time. Investigation, Formal analysis. Shaoyun Wang: Conceptualization,
The G′ and G′′ values with casein were higher than those with the chi­ Resources, Writing - review & editing, Funding acquisition.
tosan/casein complexes, chitosan, and the complexes with NaCl. The G′′
was larger than G′ throughout the adsorption, which was similar to Declaration of competing interest
former results (Figs. 5 and 6). The results were in agreement with the
previous studies (Erni, Windhab, & Fischer, 2011; Freer, Kang, Fuller, & The authors declare that they have no known competing financial
Radke, 2004; Li et al., 2016), showing that the G′′ of casein was larger interests or personal relationships that could have appeared to influence
than G′ and formed an interfacial layer like fluid. Similar results were the work reported in this paper.
also reported for isolated wheat protein, which formed a weak fluid
interface with a low interfacial modulus (Day, Xu, Lundin, & Wooster, Acknowledgments
2009). Recently, many studies had shown that the prerequisite for the
formation of a stable emulsion was not limited to the G′ of the interface, We are grateful to the National Natural Science Foundation of China
the interfacial viscosity also played important roles in the stability of the (No. U1905202), National Key R&D Program of China (No.
emulsion (Li, Xu, Lundin, & Wooster, 2009). It was worth mentioning 2016YFD0400202) for supporting this work.
that although the G′ and G′′ of chitosan/casein complexes were lower
than that of casein, the emulsion formed with chitosan/casein com­
plexes had superior stability. This could be attributed to the superior

7
F. Zhang et al. Food Hydrocolloids 111 (2021) 106211

Appendix A. Supplementary data Laplante, S., Turgeon, S. L., & Paquin, P. (2006). Emulsion-stabilizing properties of
chitosan in the presence of whey protein isolate: Effect of the mixture ratio, ionic
strength and pH. Carbohydrate Polymers, 65(4), 479–487.
Supplementary data to this article can be found online at https://doi. Li, X., Fang, Y., Al-Assaf, S., Phillips, G. O., & Jiang, F. (2012). Complexation of bovine
org/10.1016/j.foodhyd.2020.106211. serum albumin and sugar beet pectin: Stabilising oil-in-water emulsions. Journal of
Colloid and Interface Science, 388(1), 103–111.
Li, X., Fang, Y., Phillips, G. O., & Al-Assaf, S. (2013). Improved sugar beet pectin-
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