Carbohydrate Polymers 266 (2021) 118148

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Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol

Comparison of binary cress seed mucilage (CSM)/β-lactoglobulin (BLG) and

ternary CSG-BLG-Ca (calcium) complexes as emulsifiers: Interfacial
behavior and freeze-thawing stability
Afsaneh Taheri a, Mahdi Kashaninejad a, *, Ali Mohammad Tamaddon b, Seid Mahdi Jafari a
a
Department of Food Process Engineering, Faculty of Food Science and Technology, Gorgan University of Agricultural Sciences and Natural Resources, Gorgan, Iran
b
Department of Pharmaceutical Nanotechnology and Center for Nanotechnology in Drug Delivery, Shiraz University of Medical Sciences, Shiraz, Iran

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

Keywords: Protein–polysaccharide complexes often exhibit amended techno-functional characteristics when compared to
β-Lactoglobulin their individual participant biomolecules. In this study, a complex coacervation of cress seed mucilage (CSM)/
Complex Coacervation β-lactoglobulin (Blg) was used for stabilizing oil-in-water emulsions; they were characterized in terms of physical
Cress seed
properties, droplet-size distribution and microstructure. Also, a comprehensive study was carried out on inter­
Emulsion
Freeze-thawing
facial rheological responses and on the corresponding emulsion stability of different complexes. Freeze-thaw
stability of the produced emulsions which had from mixtures of CSM-Blg was also evaluated. More than the
size of droplets, interfacial rheological characteristics were associated with the properties of the adsorbed layers
and with the stability of emulsions in storage. Using the CSM-Blg-Ca ultimately resulted in emulsions that proved
stable against creaming, with no sign of phase separation over 3 weeks. These results show pro­
tein–polysaccharide complexes as appropriate emulsifiers that can make emulsion-based products resistant to
unwanted changes caused by freeze-thawing.

1. Introduction
Interactions between proteins and polysaccharides through covalent
bonding or electrostatic interactions usually play significant roles in
improving the structure and stability of emulsions, thereby highlighting
their importance in emulsion-based food industries, personal care
products and pharmaceutical applications (McClements, 2012; Mehrnia
et al., 2016). There are often two different approaches in creating
emulsions with protein-polysaccharide complexes. The first approach
involves adding an oil phase to an aqueous solution containing pro­
tein–polysaccharide complexes, followed by mixing two phases (i.e.
homogenization or sonication). The second approach involves creating
an initial emulsion first, one that is stabilized by proteins, and then
adding polysaccharides which adsorb onto the protein layer and orga­
nize a bilayer/multilayer that can increase the bridging flocculation
(Bago Rodriguez et al., 2018; Jafari et al., 2007; Taheri & Jafari, 2019b).
Generally, under appropriate conditions of formulation by the two ap­
proaches, emulsions can become more resistant to environmental
stresses as a result of adding protein–polysaccharide complexes with
multilayered interfacial membranes at the emulsion droplet surface
(Taheri & Jafari, 2019a). Environmental stresses can include thermal
treatments, freezing, drying and mechanical agitation. The degree of
stability that is imparted to emulsions, when enhanced with pro­
tein–polysaccharide complexes, usually exceeds the stability of emul­
sions in which proteins are used alone (i.e. as a single interfacial layer).
For instance, the stability of oil-in-water emulsions can be increased by
pectin-whey protein (Esfanjani et al., 2015; Faridi Esfanjani et al., 2017;
Gharehbeglou et al., 2019; Mohammadi et al., 2016), chitin nano­
crystals-β-lactoglobulin (Gülseren & Corredig, 2013), sodium caseinate-
gellan gum mixtures (Sosa-Herrera et al., 2008), maltodextrin-whey
protein (Assadpour et al., 2016a, 2016b), lactoferrin-ι-carrageenan
(Shimoni et al., 2013) and β-lactoglobulin-chitosan (Chang et al., 2018).
By common knowledge, some products and ingredients can resist
deterioration after they are repeatedly frozen and thawed (Iwanaga
et al., 2008). Termed freeze-thaw stability, this feature is technically
important when managing emulsions for food formulation. It can be
useful for extending the shelf life of products (e.g. sauces for frozen
meals) and for imparting better quality (e.g. ice cream). Nevertheless,
carrying out freeze-thaw treatments on emulsion-based products can
sometimes serve as an environmental stress and, thus, lead to adverse

* Corresponding author.
E-mail address: kashani@gau.ac.ir (M. Kashaninejad).

https://doi.org/10.1016/j.carbpol.2021.118148
Received 2 April 2021; Received in revised form 21 April 2021; Accepted 29 April 2021
Available online 5 May 2021
0144-8617/© 2021 Elsevier Ltd. All rights reserved.
A. Taheri et al.
effects such as oiling off, coalescence or partial coalescence, flocculation
and/or creaming (Degner et al., 2014). Upon freeze-thaw treatments,
the physical stability of oil-in-water emulsions (O/W) can be improved
by cryoprotectants by controlling ice crystal growth, or with interactions
between proteins and polysaccharides. This seems to be one of the most
effective approaches for obtaining emulsion-based foods that can be
resistant to freeze-thaw treatments (Degner et al., 2014; Ghosh &
Coupland, 2008). Freeze-thaw stability can be improved by protein-
polysaccharide complexes, and a few examples include relevant en­
hancements by steric stabilization in soybean-microcrystalline cellulose
(Xu et al., 2015), interfacial films with high viscoelasticity in whey
protein isolate-soybean polysaccharides (Cabezas et al., 2019) and
ovalbumin-sodium alginate (Zhang et al., 2020).
In this work, two biopolymers have been selected to stabilize oil-in-
water emulsions. They are β-lactoglobulin (Blg), a dominant component
of whey protein, and cress seed mucilage (CSM) from garden cress seed.
Blg is a compact, small globular protein, containing 162 amino acids
with molecular weights of 18.4 kDa (Ali et al., 2016). There is a struc­
tural reassembly of the protein that can partially unfold and expose
hydrophobic residues to the lipophilic phase, thereby making it an
appropriate ingredient in the stabilization of emulsions (Bos & Van
Vliet, 2001; Damodaran, 2005). CSM is a hybrid polyelectrolyte that
contains both protein and carbohydrate polymers, with a molecular
weight of about 540 kDa subunits which can vary depending on its
origin, age or purification method. In the pH value above 4.0, CSM
carries a net negative charge which can be attributed to its D-galactur­
onic acid and D-glucuronic acid residues. Protein composition and
rheological modification have been considered as reasons for the
emulsifying properties of CSM (Taheri & Razavi, 2015a, 2015b). The
combination of these two biopolymers can potentially induce a syner­
gistic effect on emulsion stability which can be examined, although this
work pursues other aims in addition to the evaluation of emulsion
stability.
In a recent work, researchers optimized the methods of creating
complex coacervation between the cress polyelectrolyte molecule (CSM)
and β-lactoglobulin (Blg) in nanoscale ranges. In aqueous solutions, Blg
proteins interacted with CSM to form either soluble or insoluble com­
plexes depending on the experimental variables such as pH and con­
centrations of Blg and CSM. The mixture consisted of a dilute solution of
Blg (0.1% w/v)-CSM (0.15% w/v) at pH 3.5, where 10 mM calcium ions
were either added or not, which eventually led to optimum samples
(Taheri et al., 2020). In this work, we extend the idea that optimized
versions of polyelectrolyte nano-complexes, based on CSM and Blg, are
capable of stabilizing corn oil in aqueous emulsions. In this study,
emulsions were stabilized by CSM-Blg and CSM-Blg-Ca complexes which
were made of aqueous polymer mixtures (pH = 3.5). The present
research involved measuring several emulsion properties (i.e. size dis­
tribution, morphology and emulsion stability) and then the impact of
freeze-thawing was evaluated on stabilized o/w emulsions that con­
tained the nano-complexes. Since the interfacial rheological evolution at
an oil/complex interface authorizes a better understanding of the
interfacial adsorption and the stabilizing mechanism of these emulsi­
fiers, diverse interfacial rheological tests were necessary for this study.
These tests comprising comprised measurements of the dynamic shear at
various deformation amplitudes and frequencies. The findings can lead
to basic information on the design of efficient nano-delivery systems in
future applications. New food-grade stabilizers can be introduced based
on polyelectrolytes Blg-CSM mixtures and they can be incorporated into
a wide range of frozen food emulsions.
2. Materials and methods
2.1. Materials
Cress seed were purchased from a local medicinal plant market. Blg
(purity 93%), CaCl2 (anhydrous), ethanol (99.5%), hydrochloric acid
2
Carbohydrate Polymers 266 (2021) 118148
(HCl) and sodium hydroxide (NaOH) were purchased from Sigma-
Aldrich (Germany). All solutions were obtained by using deionized
water from a Nanopure water system (Nanopure Infinity, USA).
2.2. Cress seed mucilage extraction and purification
Cress seed mucilage was extracted and purified according to methods
reported by Taheri et al. (2020). Briefly the water seed ratio of 30:1, pH
10 and soaking period (15 min) were considered as optimized conditions
of extraction. The slurry was extracted as mucilage and was purified by
ethanol precipitation. Mucilage was stirred for 10 min with ethanol at a
ratio of 1:3 (mucilage volume: ethanol volume). After stirring, the
mixture was centrifuged at 15,000 rpm for 3 min at 25 ◦ C. Freeze-drying
(LD Plus Alpha, Germany) was applied to dehydrate the mucilage.
2.3. Preparation of biopolymer nanocomplexes
The stock solutions of CSM and Blg were prepared by dissolving a
proper amount of CSM and Blg in deionized water to reach the final
concentration of 30 mg/mL and 20 mg/mL, respectively. CSM-Blg
complexes were synthesized by the post-blending acidification
method; after dilution, the solution of CSM was added to Blg dropwise.
The final mass ratio (Blg:CSM), total polymer concentration, and pH
were adjusted to 1:2, 25 mg/mL, and 3.5, respectively. The selection
procedure of the variables was based on our previous study (Taheri
et al., 2020). For the preparation of CSM-Blg-Ca complex, CaCl2 was
added to a group of samples after mixing the solutions, and then a final
concentration of 10 mM was reached in each solution. The CSM-Blg and
CSM-Blg-Ca mixtures were then slowly acidified in using by HCl and
NaOH so as to reach a final mixture with pH 3.5. The dispersions were
stirred for 4 h and stored for 24 h at ambient temperature prior to freeze-
drying.
2.4. Preparation of O/W emulsions
A two-step homogenization process was applied to produce the
emulsions at ambient temperature. Firstly, coarse emulsions were made
by adding corn oil (20% w/w) to aqueous dispersions of CSM-Blg and
CSM-Blg-Ca (2.0% w/v). Meanwhile, the solution (20 mL) was stirred at
15,000 rpm for 10 min using a high-speed homogenizer (IKA, T 25,
Germany). In the next step, the pre-emulsions were transferred to a
beaker and sonicated in an ultrasound homogenizer (Sonoplus HD-4200,
Germany) at 50% amplitude, with 1/3 of the standard tip length
immersed in a glass beaker (28 mm diameter) for 3 min. The tempera­
ture increase during sonication was hindered by putting the beaker in a
water-ice bath.
2.5. Freeze–thaw cycling
The freeze–thaw treatment was carried out on emulsions. The effects
of the freezing step were evaluated first. A total volume of 5 mL of each
emulsion type was frozen overnight using a freezer (− 18 ◦ C) and 15 h at
(− 30 ◦ C). Thereafter, the emulsions (previously frozen at − 18 ◦ C and
− 30 ◦ C) were thawed during 10 h at room temperature.
2.6. Emulsions droplet size and zeta-potential analysis
The droplet size, size distribution and zeta-potential (ζ) of oil drop­
lets in each emulsion were measured at 25 ◦ C by a laser-scattering
particle-size analyzer (Horiba SZ-100 analyzer, Kyoto, Japan). All
emulsions were diluted with deionized water (1:100) before analysis.
The droplet size was as the mean diameter of volume-surface. To specify
the distribution width of droplet sizes (based on volume), the ‘span’ had
to be computed first from the following formula:
A. Taheri et al.
DV,0.9 − DV,0.1
Span = (1)
DV,0.5
where Dv,0.5, Dv,0.1 and Dv,0.9 are values of the particle sizes, compared
to which 50%, 10% and 90% of the sample particles are smaller,
respectively.
2.7. Storage and creaming stability
The storage stability of each emulsion was reported by testing the
droplet size of each emulsion against different time periods (1, 8, 20, 30
days). Four milliliters of emulsion were placed in a glass test tube (with
an internal diameter of 15 mm and a height of 100 mm). These samples
were stored at 4 ◦ C and 25 ◦ C, and the movement of any creaming
boundary was measured weekly (up to 4 weeks) after the emulsion was
produced. The amount of creaming was calculated by using the
creaming index, which was calculated according to Eq. (2). Where Ht
and Hs are the total height of each emulsion and serum layer height (Hs),
respectively.
Ht
Cremaimg index = × 100 (2)
HS
2.8. Optical microscopy measurements
Optical microscopic observations of the emulsions were performed
using a Raman microscopic microscope (Horiba, Japan) to determine
the droplet size and the aggregate state of droplets. Samples were placed
on a microscope slide, covered with glass cover slips and observed using
objective lenses of 5× and 10×.
2.9. Interfacial rheology
Interfacial viscoelasticity measurements were carried out by a
rheometer equipped with a Dunoy ring (MCR 302, Anton Paar, Austria).
Regarding the liquid/liquid experiments, each nano-complex solution
had a total biopolymer concentration of 0.25% w/v. The solution was
poured into a trough, while the ring was carefully immersed in the
sample to half depth. Corn oil was poured gently into the top of the
solution by a syringe. This was to prevent the mixing of the two phases.
The interfacial rheological results were diverse and were indicated as
time-dependent evolution of the moduli (elastic (G′ ) and viscous
modulus (G′′ )), dynamic shear at different deformation amplitudes and
frequencies, along with adjusted shear deformation oscillatory runs in
which the strain amplitude and angular frequency of the oscillations are
kept constant at 1% and 1 rad/s, respectively.
2.10. Interfacial tension measurements
The interfacial tension for each Blg-CSM mixture (0.2% w/v) and the
Blg control was measured using a Lauda TD2 Tensiometer (Lauda-
Königshofen, Germany) equipped with a Du Nüoy ring (20 mm diam­
eter). Within a 40 mm diameter glass sample cup, a 20 mL biopolymer
solution was added, followed by the immersion of the Du Noüy ring and
then the addition of an upper corn oil layer (20 mL). The ring was then
pulled upward to stretch the interface to measure the maximum force
without breaking into the oil phase. Interfacial tension was calculated
from the maximum force (Fmax; units: milli-Newton; instrument mea­
sures force as mg × gravity) using the following equation (Eq. (1)):
Fmax
γ=
4πRβ
where, γ is the interfacial tension (mN/m), R is the radius of the ring (20
mm), and β is a correction factor that depends on the dimensions of the
ring and the density of the liquid involved (Lam & Nickerson, 2014).
3
Carbohydrate Polymers 266 (2021) 118148
2.11. Statistical analysis
All experiments had triplicate samples for analysis. The results were
reported as mean values and standard deviation (mean ± SD). Differ­
ences between the mean values were determined using the analysis of
variance (ANOVA) via SPSS 16.0 (SPSS Inc., Chicago, IL, USA).
3. Results and discussion
The mean droplet size was applied to measure the size of emulsions
quantitatively, considering the fact that samples were nanometer-sized.
As indicated in Fig. 1, the presence of calcium ion in the oil-water
interface influenced the droplet size of emulsion. The droplet size of
emulsion increased by calcium ion (from 164 nm to 326 nm). The
emulsion droplets were stabilized by CSM-Blg and they created uniform
spheres (Fig. 1a). On the other hand, with a CSM-Blg complex, the
surface of the corn oil droplets enabled the adsorption of more nano­
particles. This was followed by a gradual dispersion of CSM-Blg within
the emulsions, along with the production of smaller droplets (Esfanjani
et al., 2015; Linke & Drusch, 2018). The optical images of microscopy
demonstrated that droplet sizes grow when calcium ions are added to
the emulsion. In fact, big drops were observed in the images of emul­
sions where CSM-Blg-Ca was used, as compared to the use of CSM-Blg.
On the other hand, using CSM-Blg made the emulsions fluid, with
separate, small and almost monodispersed drops. By CSM-Blg-Ca, the
emulsions harbored big and slightly flocculated drops (Fig. 1b).
Storage stability is a significant factor of any products because it
specifies whether a formulation is appropriate for its planned use. The
creaming, phase separation and alterations in size distribution can be
considered as indices of storage stability. Creaming is an initial indicator
of storage stability because, parallel to creaming, coalescence and floc­
culation occur microscopically. The creaming index of CSM-Blg and
CSM-Blg-Ca emulsions were evaluated at 4 ◦ C and 25 ◦ C (Fig. 2). After
monitoring the samples for 14 days, CSM-Blg emulsions exhibited an
increase in the creaming index at 25 ◦ C (Fig. 2a) which enhanced over
time in storage (from 2.9% to 14.0%). Nonetheless, the creaming index
increased by a lesser extent at 4 ◦ C, as compared to the increase at 25 ◦ C
(from 2.3% to 7.0%) (Fig. 2a). One can conclude that a better stability is
created by CSM-Blg when emulsions are at 4 ◦ C. Herein, there is a good
agreement between the colloid characteristics of CSM-Blg aqueous
dispersion. These characteristics include surface charges (Blg = 21 ±
0.2 mV & CSM = − 68 ± 1.2 mV) and relevant emulsion properties,
while suggesting that the particles adopt various adsorption modes that
rely on their surface charges. In other words, surface charges of CSM-Blg
(− 54 ± 0.4 mV) and CSM-Blg-Ca (− 42 ± 1.1 mV) at pH ~3.0 could be
seen in relation to instability. According to Danov et al. (2006), highly
charged particles are unlikely to be precursors of stability in emulsions
due to a repulsive energy that a charged particle usually acquires in
becoming contiguous to the oil-water interface. For highly charged
particles, this force of repulsion could outweigh convective powers
during emulsification. Therefore, charged particles are likely to be
adsorbed on the interface of the oil-water blend, thereby making
emulsions unstable. Similar results were reported by De Folter et al.
(2012), indicating unstable emulsions that were stabilized by nano­
particles with high zeta potentials.
In contrast, no creaming was observed in emulsions that had been
treated with calcium ions at 4 ◦ C for 14 days. After 21 days, a slight
degree of creaming was observed (5.0%) and, thereafter, the creaming
index tended to stabilize and no more creaming occurred (Fig. 2b). The
emulsion stability showed that CSM-Blg-Ca were more capable of hin­
dering the creaming of emulsions in comparison with the capability of
CSM-Blg at 4 ◦ C and 25 ◦ C. This result could provide a background for
the viscosity enhancements in the continuous phase, thereby making
dispersed droplets move slowly. This result is in agreement with previ­
ous research by Fan et al. (2017), where adding calcium ions (9 mM) to
low-methoxylated citrus (LMP) solutions ultimately enhanced the
A. Taheri et al.
Frequency (%)
(a)
Frequency (%)
(b)
Fig. 1. Microscopic images of emulsions with added CSM-Blg complexes are taken with optical microscopy; a) CSM-Blg and b) CSM-Blg-Ca. The scale bar is 1 μm.
Fig. 2. Creaming index of emulsion prepared CSM-Blg mixtures a) 25 ◦ C and b) 4 ◦ C.
stability of emulsions modulating the system viscosity. Araiza-Calahorra
et al. (2018) reported enhancements in binding affinity between
curcumin-whey proteins in emulsions at pH 7.0 and in the presence of
10 mM CaCl2.
For a better understanding of storage stability, changes in the size of
emulsions over time were investigated. Both o/w emulsions had a nar­
row distribution (small span) and the distribution moved toward bigger
emulsion size through time (Table 1). The appearance suggests that the
fresh emulsions remain homogeneous and no phase separation was
observed for both CSM-Blg and CSM-Blg-Ca during storage (Fig. 3).
These findings are in agreement with previous results reported by Porras
4
Carbohydrate Polymers 266 (2021) 118148
Undersize (%)
Diameter (nm)
Undersize (%)
Diameter (nm)
et al. (2008) where the droplets in nano range enlarged gradually in the
absence of phase separation. This led to sedimentation which contrib­
uted to Ostwald ripening and mechanisms of coalescence. On the other
hand, bigger droplets exhibit higher coalescence rates, as compared to
small droplets at the time of storage. Whereas coalescence is less likely
to occur in the case of small droplets, medium-sized and big droplets
tend to enlarge. Nonetheless, small droplets increase in number. At the
time of storage, thus, the droplet size grows bigger because of a higher
rate of Ostwald ripening and coalescence (Capek, 2004; Jafari et al.,
2008).
The stability behavior of the emulsions, along with size, could be
A. Taheri et al. Carbohydrate Polymers 266 (2021) 118148

Table 1 complex had electrostatic repulsion with droplets. Stable emulsions

Changes in droplet size and span number in emulsions stabilized with CSM-Blg- were prepared using particles as stabilizers, demonstrating that the
Ca & CSM-Blg during 30 days at 25 ◦ C. convective power as mechanical energy input to the oil-water interface
Sample Droplet size (nm) Span is larger than the repulsive forces. Some research has reported that
CSM-Blg
particles with the smallest sizes are in a better situation to create sta­
Day 1 178 ± 10d 0.10 ± 0.02c bility (Li et al., 2014). In the current research, however, other parame­
Day 8 225 ± 13c 0.19 ± 0.03c ters such as surface chemistry and interfacial properties affected the
Day 20 363 ± 21b 0.30 ± 0.05b ability of CSM-Blg and CSM-Blg-Ca to stabilize emulsions.
Day 30 443 ± 27a 0.44 ± 0.07a
Many emulsion-based commercial food products such as sauces,
CSM-Blg-Ca mayonnaise, soups and whipped toppings can have a longer shelf life if
Day 1 345 ± 13d 0.09 ± 0.01c they are frozen. Freezing is usually followed by thawing at most stages of
Day 8 485 ± 20c 0.10 ± 0.01bc
processing and also prior to consumption. Nonetheless, poor levels of
Day 20 610 ± 31b 0.14 ± 0.02b
Day 30 824 ± 47a 0.29 ± 0.03a freeze-thaw stability characterize many food emulsions because droplets
usually coalesce as a consequence of crystallization in the oil and/or
a–d: Means followed by the same lower case in the same column are not
water phases (Thanasukarn et al., 2004). In this study, all emulsions
significantly different (P > 0.05).
were initially evaluated in terms of freeze-thaw stability by successive
acts of freezing (− 18 ◦ C, 24 h and − 30 ◦ C, 18 h) and thawing (25 ◦ C, 10
explained by spontaneous desorption. This holds true when radius r is h). The stability was gauged against creaming, coalescence and oiling
considered for spherical particles and r2 corresponds with the free- off. The hypothesis was that the synergistic effect of CSM and Blg in
energy of spontaneous desorption (Dickinson, 2010). This indicates emulsification led to a thick layer around the emulsion droplets, thereby
that the enhanced particle size of the CSM-Blg-Ca also participated in the preventing instability during freezing (Aoki et al., 2005). Regarding
enhanced physical stability of the CSM-Blg-Ca emulsion. In fact, ac­ soluble soybean polysaccharides in mixture with whey protein isolate,
cording to Coehn's rule, in a dispersion system composed of two non- Cabezas et al. (2019) reported that oil-in-water emulsions can become
conductors, a larger amount of dielectric constant in one phase is posi­ more stable against freeze-thaw due to the nature of the said mixture.
tively charged whereas the other is negatively charged (Kotsuki et al., The emulsification ability of the two components in the mixture
2016). The dielectric constant of water (78.4 F/m) is much greater than enhanced the interactions at the oil/water interface in favor of freeze-
that of corn oil (2–3 F/m). When corn oil was added to the water phase thaw stability.
during emulsification, the surfaces of the droplets were negatively All emulsions were hardly stable against oiling off. The freeze-thaw
charged. The values of zeta potential in CSM-Blg and CSM-Blg-Ca par­ treatment caused negligible change in the emulsions resistance to
ticles were − 54 mV and − 42 mV, respectively, and so the particles in a creaming. The observations intelligibly demonstrated that the CSM-Blg-

Day 1 Day 28

CSM-Blg CSM-Blg-Ca CSM-Blg

CSM-Blg-Ca

(a)

Day 1 Day 28

CSM-Blg-Ca CSM-Blg CSM-Blg-Ca CSM-Blg

(b)
Fig. 3. Visual observation of emulsion prepared CSM-Blg mixtures after 28 days storage a) 4 ◦ C and b) 25 ◦ C.

5
A. Taheri et al.
Ca complex can better assist highly-stable emulsions to take form at both
− 18 and − 30 ◦ C. After thawing at room temperature, the samples
showed that both emulsions with CSM-Blg and CSM-Blg-Ca acquire
some amount of susceptibility to the aggregation of droplets after
freezing and thawing at − 30 ◦ C. There was an increase in the average
diameter of particles from 164 nm to 669 nm by CSM-Blg and from 326
nm to 1150 nm by CSM-Blg-Ca after the freeze-thaw treatment. Since
CSM-Blg-Ca (larger particles) can better prevent re-coalescence through
the emulsifying process, resistance to creaming is improved in associa­
tion with coalescence stability. Therefore, it is reasonable to suggest that
the emulsion does not expand significantly by thawing. This is especially
true in the case of emulsions treated with CSM-Blg-Ca, whereby the
emulsion is likewise more stable (Chen et al., 2019; Jafari et al., 2008).
In this study, slow cooling (− 18 ◦ C freezer), as compared to fast cooling
(− 30 ◦ C), led to a greater stability of samples, especially because of the
relatively large CSM-Blg-Ca which increased the viscosity of the unfro­
zen aqueous phase. It reduced crystallization and maintained the quality
of samples through freeze–thaw. Tippetts and Martini (2009) reported
that large and stable crystals are more likely to form when the cooling
rate is slow, thereby resulting in higher stability of the emulsion,
particularly when samples contain smaller amounts of oil (20%, v/v).
The nature of the interfacial layer between the oil droplets and the
complexes is a significant parameter that affects the stability of emul­
sions in the freeze-thaw process and also influences indices that define
stability, e.g. creaming index (Mun et al., 2008). Thus, the characteris­
tics of interfacial oil/complexes were evaluated in this study.
Fig. 4 demonstrates the alterations of interfacial tension as a function
of adsorption time. After approximately 2 h, the graph showed an im­
mediate and significant reduction in the oil-water interfacial tension
from 23.0 to 17.6 mN/m, upon adding 0.2 (w/w)% Blg to water. This
was in agreement with the expected surface characteristics of Blg
(Bouyer et al., 2011). Whereas CSM did not indicate any immediate
considerable oil-water interfacial tension decrease upon CSM addition.
It is also worth noting that the lowest interfacial tension values recorded
for CSM alone were similar to those of Blg alone. It means that CSM
displayed good surface characteristics, but it required longer times than
Blg to reach equilibrium. Comparing the time evolution of the interfacial
tension for oil-Blg, oil-CSM, and oil-Blg-CSM dispersions at 0.2 (w/w)%,
all adsorption isotherms demonstrated similar profiles. For CSM-Blg and
CSM-Blg-Ca mixtures, interfacial behavior indicated an immediate
reduction which is similar to that calculated with Blg alone. These
findings can be related to the act that CSM is a polysaccharide and re­
quires time to be adsorbed on the interface and thus to decrease the
interfacial tension. A variation could be found when adding the CaCl2.
Fig. 4. Time evolution of the interfacial tension for oil-0.2 (w/w)% biopolymer
aqueous solutions.
6
Carbohydrate Polymers 266 (2021) 118148
At the CSM-Blg, the interfacial tension reduction seems somewhat less
extended (from 47.87 to 30.12 mN/m) at CSM-Blg compared to the
CSM-Blg-Ca (from 47.87 to 28.01 mN/m at the same concentration),
probably due to the relatively lower surface charge of the complex in the
aqueous phase which affected the surface pressure. A considerable
reduction was observed in the interfacial tension (from 30.12 to 5.22
mN/m at CSM-Blg) compared to the CSM-Blg-Ca (from 28.01 to 5.18
mN/m at the same concentration). In other words, the Blg-CSM mixtures
resulted in slightly lower interfacial tension values, accounting for a
synergy between the two emulsifiers. Dickinson (2003) also presented
that protein-polysaccharide electrostatic complexes have improved
interfacial properties compared to the single ingredients (Dickinson,
2003).
One mechanical feature of emulsions is the viscoelasticity of fluid-
fluid interfaces. It is an indicative kinetic of polymer adsorption by an
interface of oil/water and determines the strength of oil-water films
when molecules arrange and stabilize the films. According to Benjamins
and Lucassen-Reynders (1998), the interfacial viscoelastic behavior can
be considered as important as the interfacial tension value when
assessing interface characteristics, exclusively with the aim of gauging
how oil droplets are stabilized. On the other hand, the viscoelastic na­
ture of an interface can improve how stable emulsions are, while
droplets in the emulsion coalesce and the rate of film thinning is reduced
(Tambe & Sharma, 1994). In the case of CSM-Blg-Ca and CSM-Blg
(Fig. 5a), the kinetics of surface viscoelasticity and the expansion of
CSM-Blg-Ca make progress through two steps. It involves a primary and
sharp increase in G′ and G′′ , while a slower increase occurs in the second
step. This highlights the importance of processes in molecular adsorp­
tion and by an assembly between molecules at the nanocomplex/oil
interface. A similar phenomenon exists (i.e. two-step interfacial ageing)
in protein-adsorbed films and lipophilic surfactants (Opawale &
Burgess, 1998).
In contrast, CSM-Blg films can stage rapid kinetics of formation
(Fig. 5a). Obviously, a predominant elasticity exists in the case of
adsorbed layers in CSM-Blg-Ca and CSM-Blg (G′ > G′′ ). Compared with
CSM-Blg, the network structure of CSM-Blg-Ca was denser, and so the
interface storage modulus (G′ ) and interface loss modulus (G′′ ) of CSM-
Blg-Ca differed from that of CSM-Blg. As a result, its G′ indicated that a
densely packed interfacial layer is expanding. The oil-water interfacial
viscoelasticity is a determinant factor by which the stabilization per­
formance can be measured. For instance, a higher elastic interface can
reduce the level of breaking off in the oil phase (Jin et al., 2017). The
strong elastic feature of the interfacial films with CSM-Blg-Ca usually
results in a better resistance against coalescence (Romero et al., 2011)
and promotes the stability of emulsions during storage. This result is in
good agreement with previous results about the higher stability of CSM-
Blg-Ca, compared to the CSM-Blg emulsion. At the L/L interface, the
time evolution of G* can be compared with the complexes in the
adsorption films (Fig. 5b). The results suggest that complexes have film-
creating ability at the L/L interface, while G* has a greater value than
the corn/pure water interface in the absence of additives (Fig. 5b).
Layers of adsorption have interfacial features at the liquid/liquid
interfaces. To evaluate their features, amplitude sweeps had to be car­
ried out at a constant frequency of 1 rad/s (Fig. 6a) Even though
emulsifiers stabilized the structure of interfacial layers, this structure is
commonly susceptible to distortion. Thus, the amplitude sweep in CSM-
Blg-Ca and CSM-Blg films are used for evaluating how the interface can
tolerate maximum permitted shear stress, without having to annihilate
the structure. Selected frequencies determine how the amplitude sweep
can function. G′′ is smaller than G′ as an order of magnitude, and this
indicates elasticity and stability for the interfaces. CSM-Blg-Ca and CSM-
Blg film layers demonstrate linear viscoelastic patterns at deformations
of γ < 20% and γ < 5%. In fact, CSM-Blg films create interfacial networks
that are disrupted remarkably at high degrees of deformation (Rühs
et al., 2012). Angular frequencies (ω) were used (5 to 100 rad/s) for
measuring frequency-dependent-dynamics at a small strain amplitude of
A. Taheri et al. Carbohydrate Polymers 266 (2021) 118148

Fig. 5. Time dependency of the interfacial shear moduli of G′ , G′′ and G* of the complexes (CSM-Blg-Ca & CSM-Blg) at same concentration (0.5% w/w) at liquid/
liquid interfaces (T = 20 ◦ C, deformation amplitude γ 0 = 1%, frequency ω = 1 rad/s).

Fig. 6. Deformation dependence of interfacial shear moduli at a frequency of 1 rad/s (a) and frequency sweep measured at a strain amplitude γ0 = 1% (b) for
complexes (CSM-Blg-Ca & CSM-Blg).

1% (Fig. 6b). CSM-Blg and CSM-Blg-Ca make the elastic behavior

dominant by G′ > G′′ and by being strongly dependent on frequency at
the tested range. No crossover frequency was demonstrated in the case
of G′ and G′′ by complexes adsorption films anywhere in the frequencies
being tested. This suggests that the created layers have viscoelastic
properties which cannot change easily.
The CSM-Blg-Ca and CSM-Blg can stabilize emulsions which then
η (Pa.s)

exhibit interfacial shear viscosities (0.5% w/w) over an extensive

domain of shear values (10− 2 ≤ γ_ ≤ 102) (Fig. 7). They showed different
levels of viscosity and considerable shear-thinning behavior. Further­
more, the evolution of η revealed hysteresis; the amounts that were
calculated in an increasing shear-rate sweep appear to be larger in
comparison with those in a descending shear-rate sweep. This contrib­
uted to a self-disassociation or association at the interface, accompanied
by a breakdown or a reconstruction at the microstructural level (Fig. 7).
On the other hand, the films developed renewed microstructures which
are unlikely to rebound thoroughly after the ruin of interfacial films.
This phenomenon (shear-thinning along with hysteresis) is usually a
general description in some small molecular emulsifiers and in protein- Fig. 7. Steady interfacial shear viscosity of oil/CSM-Blg-Ca and oil/CSM-Blg
based films (Erni et al., 2003; Sharma et al., 2011). during ascending and descending shear rate at the liquid/liquid interfaces.
There is a general agreement about the mechanism which allows
proteins in combination with polysaccharides to develop efficiently into
emulsifiers. The oil surface is where hydrophobic protein-rich parts are

7
A. Taheri et al.
adsorbed and attached, while the aqueous solution is where hydrophilic-
carbohydrate moieties leak. Bigger sizes of particles usually cause a slow
absorption of the polysaccharide chains into the interface. After being
located at the interface, the hydrophobic amino acids are exposed when
protein parts are unfolded. In realigning themselves with the oil-phase,
these proteins extend along a viscoelastic film where the aqueous phase
has carbohydrate moieties. Consequently, a clear increase in G′ occurs in
testing for interfacial shear-rheology. Once a complex is adsorbed on the
oil surface, adsorbed molecules can improve in terms of repulsive in­
teractions because polysaccharide macromolecules exhibit electrostatic
and steric hindrance which can contribute to electrostatic stability
(Dickinson, 2003). By evaluating the interface viscoelasticity or by
measuring how much an adsorbed layer is characterized by mechanical
strength, significant information can be made regarding how stable
emulsions can become (Humblet-Hua et al., 2013). However, creating
simple relations among emulsion stability and interfacial flow behavior
does not seem to be a simplified process (Dickinson et al., 1988; Wil­
liams et al., 1997). Each adsorbed film has a quantity of interfacial-
viscoelastic reaction, in addition to rheological characteristics (i.e.
predominantly elastic or viscos modulus). These values correspond
efficiently with the correlation-emulsion stability (i.e., CSM-Blg-Ca >
CSM-Blg). The ultimate purpose of the interfacial rheological charac­
teristics is to associate the properties with adsorbed layers to the pro­
cessing and storage-behavior of actual emulsions. Arriving at this aim
needs more fundamental research where model-systems can be applied,
as in this study, and thus valued correlations can arise between bulk
stability and interfacial features.
4. Conclusions
A nanoscale (CSM-Blg and CSM-Blg-Ca) stabilized emulsion was
formulated with a narrow droplet size distribution. It had no additive
based on the combined stabilization effects of the mixed proteins and
polysaccharides. The dense interfacial layers that packaged the droplets
played an important role in stabilizing the emulsions. The results
revealed that emulsions having the CSM-Blg-Ca complex had better
physicochemical characteristics, good storage stability and creaming
than emulsions containing CSM-Blg which contributed to strong elastic
features of the interfacial films. Also, the viscosity of the continuous
phase increased parallel to the increase in CSM-Blg-Ca content. The ef­
fect of the freeze-thaw on the stability of the emulsions demonstrated
that CSM-Blg and CSM-Blg-Ca caused high levels of freeze-thaw stability
in emulsions. This mixed stabilized emulsion system can have good
potential for achieving desirable properties in functional foods.
CRediT authorship contribution statement
Afsaneh Taheri: Conceptualization, Formal analysis, Investigation,
Methodology, Writing – original draft. Mahdi Kashaninejad: Funding
acquisition, Formal analysis, Project administration, Resources, Super­
vision, Validation, Writing – review & editing. Ali Mohammad Tam­
addon: Project administration, Resources, Supervision, Validation,
Writing – review & editing. Seid Mahdi Jafari: Project administration,
Supervision, Visualization, Writing – review & editing.
Acknowledgments
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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