International Journal of Biological Macromolecules 147 (2020) 595–606

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Structuring oil-in-water emulsion by forming egg yolk/alginate

complexes: Their potential application in fabricating low-fat
mayonnaise-like emulsion gels and redispersible solid emulsions
Xi Yang a, Anqi Li a, Wenyan Yu a, Xiuxiu Li a, Lijun Sun a,c, Jia Xue a,b,⁎, Yurong Guo a,d,⁎
a
College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi'an 710062, China
b
Key Laboratory of Urban Agriculture, Ministry of Agriculture, Shanghai 200240, China
c
College of Food Science and Engineering, Northwest A & F University, Yangling, China
d
National Research & Development Center of Apple Processing Technology, China

a r t i c l e i n f o a b s t r a c t

Article history: Overconsumption of fat-rich foods has caused increasing incidences of human chronic diseases. Thus, it is prom-
Received 7 November 2019 ising to fabricate reduced-fat foods, but the reduction in fat content often causes undesired textural changes in
Received in revised form 30 December 2019 foods. In this study, we showed a facile method to structure oil-in-water emulsions via the electrostatic complex-
Accepted 10 January 2020
ation between alginate (Alg) and egg yolk proteins at acid pHs (lower than pH 5.0), and the resulting emulsion
Available online 11 January 2020
gels displayed significant viscoelasticity, thixotropy and plasticity, which are comparable to a full-fat mayonnaise
Keywords:
product with 75% oil content. Besides, the textural attributes of the structured emulsion gels can be tailored by
Emulsion gels simply altering the addition amount of vinegar, a commonly used ingredient in fabricating mayonnaise-like
Alginate products to confer good sensory profile. After lyophilization, we obtained dry emulsion gels with a high oil con-
Egg yolk tent (~75%), and it was found that the dried gel prepared at pH 5.8 showed better dispersibility than at other pHs,
Complexation highlighting the potential application prospect of the gels to be developed as a carrier for loading functional oil
Rheological properties soluble compounds.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction
Evidences are growing that overconsumption of high fat contents of
foods is correlated with the increasing incidences of some chronic dis-
eases such as obesity, diabetes and coronary heart diseases [1,2]. There-
fore, there is an urgent need in the food industry to develop reduced-fat
foods with comparable sensory profiles to their full-fat counterparts.
However, it is a challenging task to achieve such a purpose, because
fat commonly plays multiple roles in real food systems, e.g., conferring
desired mouthfeel and favor profiles. Moreover, in some cases, fat also
functions as structuring agent to affect food textures, especially in fabri-
cating emulsion-related foods. When fat content is reduced, the textural
attributes, flavor profiles, mouthfeel as well as the optical properties of
the foods are inevitably changed. At present, hydrocolloids including
polysaccharides and some proteins have been increasingly used to par-
tially replace fat droplets to obtain reduced-fat products while main-
taining textures similar to those of the original products [3].
Mayonnaise is basically an oil-in-water emulsion, which can be pre-
pared by mixing egg yolk, vinegar, oil, and spices [4]. Due to good flavor
⁎ Corresponding authors at: Shaanxi Normal University, China.
E-mail addresses: cathyxue0727@snnu.edu.cn (J. Xue), Yrguo730@snnu.edu.cn
(Y. Guo).
and convenience, mayonnaise has become a popular sauce item around
the world. However, the traditionally made mayonnaises contain
70–80% of fat, which increases the public's concerns on overconsump-
tion of fats. So far, several strategies have been proposed to fabricate
low-fat mayonnaise-like products. Based on the structuring mecha-
nisms, these strategies can be classified into (i) structuring edible oil
by small molecular gelators or by ternary protein/polyphenol/polysac-
charide complexes [5,6]; (ii) preparing high internal phase emulsions
[7–11]; (iii) replacing fat droplets with micro-gel particles or other fat
substitutes [12,13]; and (iv) dispersing emulsified oil droplets into the
gelling continuous phases that possess rheological characteristics simi-
lar to mayonnaise products [14,15]. In strategy (i), fabrication of the ter-
nary complexes is not easily to be achieved, which requires intricate
control of the preparation conditions; in strategy (ii), although high in-
ternal phase can simulate the textural attributes of mayonnaise prod-
ucts, high internal phase emulsion still requires a relatively high oil
content. Therefore, strategy (iii) and (iv) are more promising to be
adopted to fabricate low-fat mayonnaise-like products [16]. In terms
of textures, mayonnaise products possess significant viscoelasticity,
thixotropy and stackability/plasticity, which means that mayonnaises
are solid-like and spreadable. Therefore, the principle of fabricating
low-fat mayonnaise-like products should necessitate the three criteria:
thixotropy, viscoelasticity and stackability [17].

https://doi.org/10.1016/j.ijbiomac.2020.01.106
0141-8130/© 2020 Elsevier B.V. All rights reserved.
596 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606
Alginate (Alg) is the main structural component in marine brown
(Phaeophyceae), and it is composed of linear copolymers of (1 → 4)-
linked β-D-mannuronic acid (M) and α-L-guluronic acid (G) residues
in varying sequences [18]. At sufficiently high Ca2+ concentration, Alg
can form a cohesive and firm gel [19–21]; in comparison, when Ca2+
concentration is relatively low, Alg only form thixotropic solution in-
stead of a self-supporting gel [22,23]. Based on these properties, the po-
tential application of Alg in fabricating low-fat mayonnaise-like
products has been investigated in our previous works [16,17]. Although
the textural properties of the fabricated products are similar to full-fat
mayonnaise products, the Ca2+ concentration is found difficult to ma-
nipulate. For example, excessive Ca2+ ions easily lead to formation of
pre-gel particles via rapidly combining Alg chains, whereas insufficient
Ca2+ did not yield the desired textural properties.
In the present work, we have proposed a novel and facile methodol-
ogy to produce low-fat mayonnaise-like emulsion gels. Given that vine-
gar is commonly added in mayonnaise products to confer desirable
flavor profiles, we hypothesized that at appropriate pH conditions (vin-
egar addition), Alg can bind with egg yolk proteins to form electrostatic
complexes, which may be able to structure the overall system [24,25]. In
this system, the textural attributes can be easily tuned by changing the
addition amount of vinegar, provided that the vinegar addition is ac-
ceptable at sensory levels. Therefore, in this study the effects of vinegar
addition on the rheological properties of the prepared emulsion gels
were systematically investigated by performing rheological tests and a
combination of confocal and scanning electron microscopy techniques.
It is expected that our results can provide a facile strategy for fabricating
low-fat mayonnaise-like products, which may help enrich the method-
ologies for developing gel-related functional foods.
2. Materials and methods
2.1. Materials
Alginate (Alg) with an average molecular mass of 200 kDa and the
M/G ratio of ~1.56 was provided by Yuanye biological technology Co.,
Ltd. (Shanghai, China), and used without further treatments. Hen eggs,
edible rapeseed oil and white vinegar were purchased from a local su-
permarket. Other chemical reagents including citric acid, citrate sodium,
Nile red, Nile blue A, 1, 2-propylene glycol, etc., were of analytical grade
and also provided by Yuanye biological technology Co., Ltd. All water
used was deionized water.
2.2. Visual observations of the mixed solution of egg yolk and Alg at different
pHs
To investigate the complexation behavior of Alg and egg yolk at dif-
ferent pHs, we prepared a series of dilute mixed solutions consisting of
Alg and egg yolk. Briefly, 0.1 M citric acid and 0.1 M citrate sodium were
used to prepare the buffer solutions with gradient pH values
(pH 3.0–5.8). Subsequently, 0.5 mL egg yolk was added in the buffer so-
lutions (40 mL) to be uniformly dispersed under vortex oscillation. Af-
terwards, 1 mL 1% Alg solution (w/v) was added in the egg yolk-
contained buffer solutions, followed by vigorous shaking so as to uni-
formly mix Alg and egg yolk. After the solutions were kept at room tem-
perature for 2 h, the aggregations of the Alg and egg yolk can be
observed. In the meantime, the buffer solutions containing only
0.5 mL egg yolk (without Alg added) were also prepared and served
as a control.
2.3. Zeta potential ζ
0.5% Alg solution was prepared by dissolving 0.5 g Alg powder in
100 mL of citric acid and citrate sodium buffer at four pH gradients
(i.e., pH = 3.0, 3.8, 5.0 and 5.8, respectively). Similarly, 2.0% egg yolk dis-
persion was also prepared by dispersing 2.0 egg yolk in 100 mL of citric
acid and citrate sodium buffers. After vigorous shaking of the dispersion,
approximately 3 mL of the solution was poured into a quartz cell to be
determined by a Zetasizer (ZS-90, Malvern, UK) to obtain the zeta po-
tential (ζ).
2.4. Preparation of the mixed Alg/egg yolk systems
We further prepared the mixed Alg and egg yolk systems with a
higher Alg and egg yolk concentration and at different pHs. In brief,
0.1 M citric acid and 0.1 M citrate sodium were used to prepare a class
of buffer solutions with gradient pHs (i.e., pH 3.0, 3.8, 5.0, 5.8). Subse-
quently, 2.0% Alg solution (w/v) was prepared by dispersing Alg powder
in the buffer solutions to be hydrated under magnetic stirring for 12 h.
To avoid decomposition of Alg under acid pHs, all hydration was per-
formed at room temperature (~20 °C). Afterwards, 50 mL 2% of Alg so-
lution was poured into a 100 mL beaker, followed by mixing with
different amounts of egg yolk, with each system containing 1, 5 and
10 mL of egg yolk, respectively. In the meantime, pure Alg solutions at
different pHs served as control. It was clearly observed that only the sys-
tems at pH 3 and 3.8 and with 5 and 10 g egg yolk added could exhibit
significant stackability and solid-like property.
2.5. CLSM observations of the aggregates of Alg and egg yolk
A confocal laser scanning microscope (CLSM, FV1200, Olympus Cor-
poration, Japan) was applied to visualize the morphologies of Alg and
egg yolk aggregates. Firstly, ~ 0.2% Nile red staining solution was pre-
pared by dissolving 20 mg Nile red powder in 10 mL 1, 2-propylene gly-
col and stored at 4 °C until use. After the egg yolk was mixed with the
Alg solution, 100 μL of Nile red staining solution was subsequently
added, followed by vigorous stirring. When observed, a smear of the
sample was loaded onto a glass slide, covered by a cover slip. Subse-
quently, the sample was imaged by operating the CLSM under fluores-
cence mode, and the excitation wave was set at 559 nm.
2.6. Scanning electronic scope (SEM) observations
An Environmental Scanning Electron Microscope (ESEM, Quanta
200, FEI, USA) was adopted to reflect the aggregate morphologies of
the mixed Alg/egg yolk systems. All samples were prepared according
to the method described in section 2.4. Subsequently, the samples
were immersed in liquid nitrogen to be rapidly frozen, followed by ly-
ophilization for 48 h until complete dehydration. Afterwards, the lyoph-
ilized samples were fractured and the fracture surfaces were sprayed
with gold powder. The surface morphology was then imaged at high
vacuum mode and the HV was 20.0 KV.
2.7. Rheological properties of the mixed Alg/egg yolk systems
As described earlier, only the mixed systems prepared at pH 3 and
3.8 and with 5 and 10 g egg yolk added could exhibit significant solid-
like property, and therefore, the four samples were selected to perform
rheological tests. A stress-controlled rheometer (AR-G2, TA Instru-
ments, USA) equipped with a rough-faced parallel plate with 20 mm di-
ameter and 1 mm of parallel plate gap was employed to perform the
rheological tests. After loading 3 mL of the sample onto the sample
stage, the apparent viscosity was recorded within 0.1–500 s−1 of
shear rate range, during which the temperature was kept at 20 °C. To re-
flect the viscoelasticity, we also perform the creep-recovery tests. Ac-
cording to the stress sweeps results, the creep-recovery tests were
performed with 10 Pa stress imposed (within LVR). After the sample
was held at 20 °C for 10 min to relax any extra stress, 10 Pa stress was
instantly imposed on the sample for 600 s, during which the compliance
(J, Pa−1) was recorded as a function of time. Immediately, the compli-
ance was recorded for another period of 600 s after immediately remov-
ing the applied stress.
 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606
Furthermore, the thixotropic properties of the mixed Alg/egg yolk
systems were also evaluated by two continuous steps of oscillatory
strains: 1000% and 1% strains were applied to break the structure of
the mixed systems and measure their recovery property, respectively
[26,27]. A thixotropic test consisted of five cycles and all rheological
tests were performed at 20 °C.
2.8. Preparation and characterization of mayonnaise-like emulsion gels
The low-fat mayonnaise-like emulsion gels were prepared by simply
mixing Alg solution with egg yolk-based emulsions, followed by addi-
tion of different amounts of vinegar to induce gel formation. Briefly,
4.0% Alg stocking solution (w/v) was prepared by dispersing Alg powder
in deionized water to be hydrated. Then, 30 mL of rapeseed oil was
added into 10 mL egg yolk drop by drop under vigorous stirring to pre-
pare emulsion. After the emulsion was mixed with 50 mL of 4% Alg so-
lution, 2 g salt, 2 g sugar and different amounts of white vinegar (2, 4, 6,
8, 10 mL) were added into the matrix in sequence, followed by stirring.
Deionized water was used to compensate the volume of the emulsion
gels to 100 mL. Therefore, the final mayonnaise-like emulsion gels had
a volume of approximately 100 mL and contained 2 g Alg, 2 g salt, 2 g
sugar, 10 g egg yolk and 30 mL rapeseed oil [17]. In the meantime, a
full-fat mayonnaise product containing 10 g egg yolk, 75% of rapeseed
oil, 4 mL vinegar, 2.0 g salt, and 2.0 g sugar was prepared by a similar
procedure and served as a control [8,28].
To assess the stability of the emulsion gels, all prepared samples
were subjected to heating treatment at 100 °C for 30 min and one
freeze-thaw cycle (freezing at −18 °C for 12 h followed by thawing at
room temperature), respectively. Subsequently, to reflect the micro-
structures of the prepared low-fat mayonnaise products, we adopted
Nile red and Nile blue A to stain the oil droplets and the proteins aggre-
gates in the products, respectively. In brief, ~0.2% (w/v) of Nile blue A
staining solution was prepared by dissolving 20 mg Nile blue A powder
in 10 mL deionized water. Similarly, ~0.2% (w/v) of Nile red staining so-
lution was also prepared by dissolving 20 mg Nile red powder in 10 mL
1, 2-propylene glycol. Subsequently, 0.5 mL Nile blue A staining solution
was mixed with 10 g egg yolk to be uniformly stirred and left for 2 h to
ensure satisfactory staining. Similarly, 0.5 mL Nile red solution was
mixed with 30 mL rapeseed oil to be homogeneously stirred. After
that, the low-fat mayonnaise-like emulsion gels were prepared accord-
ing to the method described above. When imaging, a smear of the emul-
sion gels was loaded onto a glass slide, followed by covering with a
cover slip. Then, the sample was imaged by operating the CLSM under
the bright field mode, fluorescence mode at the excitation wavelength
of 488 and 603 nm, respectively. All pictures were obtained at a magni-
fication of 100×.
2.9. Sensory evaluation
To evaluate the sensory attributes, we perform sensory evaluation
experiment of the prepared low-fat mayonnaise products according to
the method described elsewhere [8,28]. In brief, after preparing the
samples as described in section 2.8 and leaving them at room tempera-
ture for 24 h, 15 trained panel consisting of eight males and seven fe-
males who aged 18–35 were invited to score the samples in terms of
their appearance, flavor, stickiness, smoothness, and overall acceptabil-
ity. 9 point stands for the most liked score and 1 being the most disliked
score.
2.10. Redispersibility of the emulsion gels
The emulsion gels were prepared at four pH gradients (i.e., pH 3.0,
3.8, 5.0 and 5.8). Briefly, 0.1 M citric acid and 0.1 M citrate sodium
were used to prepare the buffer solutions with designed pH values.
Alg powder was subsequently dissolved in the buffer solutions to be hy-
drated at room temperature for 12 h to achieve 2% of concentration.
597
Then, 20 mL of egg yolk was mixed with 30 mL rapeseed oil to be man-
ually stirred to prepare emulsion, after which the emulsion was added
in 50 mL 2% Alg solution. Then, the matrix was vigorously stirred to
achieve uniform mixing, followed by lyophilization for 48 h until com-
plete dry. Because the fresh emulsion gels possessed 100 mL of volume,
1 g Alg, 20 g egg yolk and 30 mL rapeseed oil, after lyophilization, the
dried gels had an estimated oil percentage of ~75% (w/w). When ana-
lyzed, 1 g of the dried gel was added into a vial containing 10 mL deion-
ized water and the vial was held for 10 min to completely wet the gels.
Subsequently, the vial was moderately shaken to disperse the emulsion
gels. The oil droplet morphology in the dispersed emulsion was imaged
according to the protocol described in section 2.8 (under the bright field
mode). For comparison, the freshly prepared emulsion gels acted as a
control.
2.11. Statistical analysis
All measurements were performed in triplicate. One-way analysis of
variance (ANOVA) was performed by SPSS statistics 13.0 (SPSS Inc., Chi-
cago, USA). Differences were considered significant between the sam-
ples at P b .05.
3. Results
3.1. Electrostatic complexation of Alg and egg yolk
It is well established that polysaccharides, especially negatively
charged polysaccharides, can form electrostatic complex with proteins,
provided that the pH is below the pKa of the proteins [29]. Egg yolk is an
essential component for fabricating mayonnaise products and it con-
tains abundant proteins. Therefore, it is theoretically feasible to strength
mayonnaise structure by producing polysaccharide/protein complexes.
We firstly monitored the complexation behavior of mixed Alg and egg
yolk system at a wide pH range (i.e., 3.0–5.8). It was found from
Fig. 1A that the pure egg yolk can be well dispersed in the buffer solu-
tion, and at pH 5.0, slight precipitation was observed after holding the
dispersion for 2 h, which may be because egg yolk proteins have an iso-
electric point at around this pH, leading to decreased solubility. At the
pH lower than 5.0, egg yolk proteins could keep good dispersibility.
However, after adding Alg, the mixed Alg and egg yolk systems were
shown to form significant aggregates (Fig. 1A). This can be ascribed to
electrostatic attraction between Alg and egg yolk proteins, as confirmed
by subsequent zeta potential determination. As shown in Fig. 1B, at
pH 3.0–5.8, Alg molecules carried negative charges, whereas egg yolk
proteins carried positive charges when the pH was lower than 5.0.
When increasing Alg and egg yolk concentrations, it was found that
the mixed systems could yield interesting rheological properties, such
as viscoelasticity, thixotropy, and stackability. These properties may im-
part the potential application value to the systems as a template for fab-
ricating low-fat mayonnaise-like emulsion gels. Therefore, we
characterized the rheological properties of the mixed Alg and egg yolk
systems. It was found that at pH 3.0 and 3.8, the mixture could display
solid-like properties; in comparison, at pH 5.0 and 5.8, the mixed sys-
tems still behaved like viscous fluids (Fig. 1C). Then, microscopic obser-
vations including CLSM and SEM were made in order to describe the
ultrastructure of the mixed systems [30]. By CLSM, labelling the egg
yolk proteins with Nile red dye allowed us to localize the proteins in
the mixture. The stained proteins should appear as clear in the CLSM
pictures owing to the fluorescence of the Nile red, while dark areas
should correspond to zones devoid of proteins. As shown in Fig. 1D, at
pH 3.0 and 3.8, Alg could bind with egg yolk proteins to form complexes,
as indicated by red sheet-like structure. However, at pH 5.0 and 5.8, no
complexes were found; instead, egg yolk proteins were uniformly dis-
persed in the mixture system. Moreover, SEM was also adopted to re-
veal the micro-morphologies of the mixed systems. As seen from
Fig. 2, when the pH was at 5.0 and 5.8, Alg formed ordered, fiber-like
598 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606
Fig. 1. Aggregation behavior of egg yolk and Alg at different pH values. A) Visual observations; B) Zeta potential; C) Appearance of aggregates of egg yolk and Alg; and D) CLSM images of
aggregates of egg yolk and Alg.
structure, which was undisrupted by egg yolk addition, suggesting no
significant complexation occurring between Alg and egg yolk proteins.
However, at pH 3.8 and 3.0, it was found that at low concentrations of
egg yolk addition, Alg could form ordered and fiber-like structure,
whereas with increasing egg yolk addition, the ordered Alg structure
was disrupted. This can be understood by the fact that at pH 3.0 and
3.8, egg yolk proteins bound with Alg to form complexes, leading to
the disruption of Alg chains ordering during lyophilization. It is worth
noting that, the observed ordered, fiber-like structure of the pure Alg
was not very significant at pH 3.0, which may be due to the fact that
low pH caused a more compact conformation of Alg, a factor unfavor-
able for Alg ordering during lyophilization.
Furthermore, as mentioned above, only the mixtures prepared at
pH 3.0 and 3.8 exhibited significant solid-like properties (Fig. 1C), and
therefore we characterized their rheological properties so as to investi-
gate whether the mixed Alg and egg yolk systems possess the possibility
for fabricating low-fat mayonnaise like emulsion gels. As shown in
Fig. 3A, all samples exhibited very significant shear-thinning phenome-
non. At the same level of egg yolk addition, the mixture had higher ap-
parent viscosity at pH 3.8 than at pH 3.0, which may be attributed to the
formation of more significant aggregates. In addition, at a fixed Alg con-
centration, increasing egg yolk addition also yielded higher apparent
shearing viscosity. Besides, the mixture systems exhibited typical char-
acteristics of viscoelastic materials, as indicated by their creep-recovery
curves (Fig. 3B). Then, thixotropy tests were also performed to investi-
gate the spreadable properties of the mixture systems. As observed
from Fig. 3C&D, during five cycles of oscillatory shearing steps, all sam-
ples exhibited good recovery properties; after 1000% strain was im-
posed to disrupt the sample structure, the systems could almost
instantly recovery their original moduli values. These results indicate
that the mixed Alg and egg yolk systems possessed desired
viscoelasticity, thixotropy and plasticity, demonstrating their potential
application prospect in producing low-fat mayonnaise-like emulsion
gels.
3.2. Fabrication and characterization of low-fat mayonnaise-like emulsion
gels
In this section, at a fixed volume of 100 mL, we prepared a class of
emulsion gels which contained 2 g Alg, 2 g salt, 2 g sugar, 10 g egg
yolk, 30 mL rapeseed oil and varying volumes of white vinegar. The ap-
pearance and oil droplet morphologies of the emulsion gels are shown
in Fig. 4. As reflected, with increasing vinegar addition, the emulsion
gels exhibited strengthened structure, whereas the oil droplet morphol-
ogy was similar, indicating homogeneous emulsification. It was found
that after heating at 100 °C for 30 min, all emulsion gels exhibited signif-
icant oil droplet coalescence. At 0 mL of vinegar addition, the oil droplets
had undergone very significant coalescence, but at 2 mL and 4 mL vine-
gar addition, the oil droplet coalescence was not very pronounced. As
the vinegar addition further increased, the oil droplet coalescence be-
came increasingly significant. This can be ascribed to denaturation of
egg yolk protein at high temperatures. Without vinegar addition, the
emulsion gel had relatively weak structure, and on heating, protein de-
naturation led to decreased emulsion stability, therefore causing oil
droplet coalescence. At moderate vinegar addition (e.g., 2 mL and
4 mL), the emulsion gel had intensified structure, which could hinder
oil droplets from coalescence. Whereas, at higher levels of vinegar addi-
tion, a larger amount of egg yolk protein took part in the complexation
with Alg, leading to less protein participating in stabilizing oil droplets.
Therefore, the emulsion may show decreased stability. For comparative
analysis, we also prepared a full fat mayonnaise product which
contained 10 g egg yolk, 75 mL of rapeseed oil, 4 mL vinegar, 2.0 g
 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606 599

Fig. 2. SEM images of mixed Alg and egg yolk at different pH values.

salt, and 2.0 g sugar. It was found that both heating and freeze-thaw
treatment led to significant oil droplet coalescence, with heating treat-
ment causing a more pronounced emulsion destabilization, suggesting
that denaturation of egg yolk protein was the main factor governing
the emulsion destabilization. Different from the full-fat mayonnaise
product, however, all prepared emulsion gels displayed acceptable
freeze-thaw stability, with no significant oil droplet coalescence ob-
served after one freeze-thaw cycle (Fig. 4).
Furthermore, to better investigate the micro-structures of the pre-
pared low-fat mayonnaise-like emulsion gels, we adopted Nile red
and Nile blue A to dye the emulsified oil droplets and the egg yolk
proteins, respectively. Under fluorescence mode, the oil droplets should
appear as red color, and the stained proteins should appear as green
color, which allow us to locate the oil droplets and the proteins in the
emulsion gels. As shown in Fig. 5, all prepared low-fat mayonnaise-
like emulsion gels showed significantly flocculated oil droplets and pro-
tein aggregates, which can be explained as inadequate mixing during
the sample preparation process. According to the preparation strategy,
emulsion was firstly prepared and then mixed with Alg solution,
followed by adding vinegar to induce the complexation between egg
yolk proteins and Alg. During the procedure, vigorous stirring was per-
formed to uniformly mix the matrix. Despite this, however, it was
600 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606
Fig. 3. Rheological tests of mixed Alg and egg yolk systems at pH 3 and 3.8 and with different egg yolk addition, respectively. (A) Shear viscosity; (B) creep-recovery curves; (C) thixotropy;
and (D) phase angle changes during thixotropy tests.
expected that perfect mixing was difficult to achieve. Indeed, we ob-
served the significant green regions in the emulsion gel without vinegar
addition, where the complexation of Alg and egg yolk proteins was ex-
pected not to occur, because the pH of the mixture was determined to
be 6.55 at 0 mL of vinegar addition. With increasing vinegar addition,
however, the green region in the CLSM pictures became more signifi-
cant and began to span the whole picture, suggesting that more protein
aggregates were formed, which is due to the complexation of Alg and
egg yolk proteins. In addition, it is worth nothing that, freeze-thaw
treatment did not significantly disrupt the emulsion gel structure,
whereas heating exerted a significant impact, leading to marked oil
droplet coalescence. Specially, for the prepared high fat mayonnaise
(control), we did not obtain very clear CLSM images that can reflect
the oil droplet morphology. This is due to the fact that high fat mayon-
naise contains a very high oil content (75%), allowing the emulsion gel
to contain numerous compactly packed oil droplets, which obscured
the oil-water interface in the CLSM images. After freeze-thaw and
heating treatments, the high fat mayonnaise displayed significantly ag-
gregated oil droplets and proteins, indicating inferior freeze-thaw and
heating stability.
To reflect whether the prepared low-fat mayonnaise-like emulsion
gels showed the desired rheological properties, we further character-
ized the emulsion gels in terms of their shearing viscosity, viscoelastic-
ity, and thixotropy. As shown in Fig. 6, with increasing vinegar addition,
the emulsion gel showed higher shearing viscosity, and the shear thin-
ning phenomenon was also more pronounced. Besides, the storage
modulus G' and loss modulus G" of all emulsion gels displayed slightly
frequency-dependent behavior, and with increasing vinegar addition,
the frequency-dependence decreased, indicating that the gel structure
was strengthened. Subsequent thixotropy tests suggested that the
emulsion gels also exhibited significant thixotropic properties, with
10 mL vinegar addition yielding the highest thixotropy. For high fat
mayonnaise, the apparent viscosity, frequency-dependent behavior
and thixotropic properties were very similar to those of the low-fat
mayonnaise products at 4–6 mL vinegar addition. Furthermore, we
also compared their viscoelasticity, and it was observed that high fat
mayonnaise showed the most closed creep-recovery curve to that of
the low-fat mayonnaise product with 6 mL vinegar addition (Fig. 7).
In the subsequent sensory evaluation experiment, it was found that
the low-fat mayonnaise products with 2–6 mL vinegar addition pos-
sessed higher overall sensory scores than high fat mayonnaise
(Table 1), demonstrating their acceptability.
3.3. Redispersibility of fabricated Alg and egg yolk solid emulsions
In this section, we are inspired by the fact that sipping a small
amount of water can instantly alleviate the choking caused by cooked
egg yolk, which may suggest that egg yolk proteins are ready to disperse
in water. Therefore, we fabricated the solid emulsions by lyophilizing
the Alg and egg yolk emulsion gels. At a fixed total volume of 100 mL,
the emulsion gels containing 1 g Alg, 20 g egg yolk and 30 mL rapeseed
oil were prepared at four pH conditions (i.e., pH 3.0, 3.8, 5.0 & 5.8, re-
spectively). After lyophilization, the dried emulsion gels contained a
high oil content, which was calculated to be ~75%. All emulsion gels
did not show any oil leakage during lyophilization. Subsequent SEM ob-
servations revealed that the oil droplets were uniformly dispersed in the
fracture surfaces of the samples, as indicated by the black shadows in
the SEM images (Fig. 8). Especially, at pH 5.8, the sample exhibited sig-
nificant porous structures. In comparison, at pH 3.0 and 3.8, the samples
featured by significantly aggregated structures. Then, the dried gels
were immersed in deionized water to be completely wetted, after
which the vials were moderately shaken to disperse the gels (Fig. 9A).
 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606 601

Fig. 4. Appearance of the prepared low-fat mayonnaise-like emulsion gels and the corresponding oil droplet morphologies.
602 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606

Fig. 5. CLSM images of the stained low-fat mayonnaise-like emulsion gels.

It was found that the dried gels prepared at pH 5.0 and 5.8 exhibited bet-
ter dispersibility than at pH 3.0 and 3.8, characterized by shorter time
requirement to be completely dispersed. Despite this, within 10 min,
all the four samples could be well dispersed. However, after holding
the dispersions for a short period (~1 h), significant layer separation
was observed for the dispersions prepared at pH 3.0 & 3.8, whereas
the dispersions at pH 5.0 & 5.8 were relatively stable. After dispersion,
we further observed the oil droplet morphology in the solution. As
reflected in Fig. 9B, the oil droplets showed coalescence after re-
dispersing in water, but the oil droplet coalescence at pH 5.8 was
relatively insignificant. Because at pH 3.0 and 3.8, the re-dispersed
emulsion showed poor stability, and therefore we did not characterize
the oil droplet morphologies at the two pH conditions.
The dispersibility can be explained by the structuring mechanism of
the dried emulsion gels. As described above, at pH 3.0 and 3.8, the egg
yolk dispersion carried positive charges whereas Alg carried negative
charges (Fig. 1B), and the electrostatic attraction between Alg and egg
yolk was therefore significant, leading to electrostatic complexation be-
tween Alg and egg yolk proteins. When dispersing the dried gels in
water, the Alg and egg yolk protein complexes could not be well
 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606 603

Fig. 6. Rheological tests of the low-fat mayonnaise-like emulsion gels. (A) Shear viscosity; (B) frequency sweep results; (C) thixotropy tests; and (D) phase angle changes during thixotropy
tests.

dispersed, causing separated two layers, with the upper layer enriched
the complexes and the lower layers enriched water (Fig. 9A). However,
at pH 5.0 & 5.8, where no electrostatic complexation occurred between
Alg and egg yolk proteins, when dispersing the dried gels in water, the
Alg and egg yolk could be easily dispersed, imparting the gels good
redispersibility.
4. Discussion
In the present work, we developed a novel strategy to structure the
emulsion gel system, which is based on the complexation of egg yolk
protein and Alg (Fig. 1). When the pH value was lower than 5.0, the
complexation significantly occurred, which can be attributed to the
electrostatic attraction between egg yolk protein and Alg (Fig. 1B). As
observed, the zeta potential of the Alg solution did not significantly fluc-
tuate at the pH range 3.0–5.8, whereas with decreasing pH, the egg yolk
proteins carried more positive charges, implying a more pronounced
electrostatic complexation. As expected, at pH 3.0 and 3.8, when the
Alg and egg yolk concentrations were further increased, the complex
systems showed significant viscoelasticity, thixotropy and stackability,
but the complex systems at pH 5.0 and 5.8 remained viscous fluids
(Fig. 1C). Therefore, the results demonstrate the complexation between
Alg and egg yolk proteins has the potential application value of fabricat-
ing low-fat mayonnaise-like products.
Based on this strategy, a class of emulsion gels were prepared by
mixing egg yolk, rapeseed oil, salt, sugar and Alg solutions, followed
by addition of vinegar to induce the structuring of the mixed systems.

Table 1
Sensory evaluation of the prepared low-fat mayonnaise products.

Sample Flavor Stickiness Smoothness Acceptability

Control 6.27 ± 1.0328bc 6.53 ± 0.7432bc 6.13 ± 0.7432bc 5.93 ± 0.7988b

(high
fat)
0 mL 6.33 ± 0.7237bc 5.87 ± 1.1872c 5.67 ± 0.8997bc 5.4 ± 0.7368bc
2 mL 6.8 ± 0.9411ab 6.53 ± 0.9155bc 6.53 ± 0.8338ab 6.87 ± 0.6399a
4 mL 7.07 ± 0.7988a 7.33 ± 0.7237a 7.2 ± 0.6761a 6.93 ± 0.8837a
6 mL 6.27 ± 0.8997bc 6.93 ± 0.9612ab 6.33 ± 0.8165ab 6.01 ± 0.6547b
8 mL 6.07 ± 0.7037c 6.07 ± 1.0328c 5.87 ± 0.9155bc 5.6 ± 0.7368bc
10 mL 4.73 ± 1.0328d 5.87 ± 0.9155c 5.4 ± 0.8281c 4.93 ± 0.5936c

Note: Data in each column and labeled with different superscripts are significantly differ-
Fig. 7. Creep-recovery curves of the prepared low-fat mayonnaise like emulsion gels. ent (P b 0.05).
604 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606
Fig. 8. SEM images of the lyophilized emulsion gels prepared at different pH values. (A) pH 3.0; (B) pH 3.8; (C) pH 5.0; and (D): pH 5.8.
To better reflect the effects of vinegar addition on the rheological prop-
erties of the emulsion gels, we only altered the addition amount of vin-
egar, with both the oil content (30%, v/v) and the addition amounts of
other ingredients such as sugar, salt and Alg unchanged [31]. Indeed,
vinegar addition induced a strengthened structure of the emulsion
gels, with a higher vinegar addition yielding a better strengthening ef-
fect (Figs. 4–7). We found that the pH values of the prepared low-fat
mayonnaise products were 6.55 (0 mL vinegar), 4.94 (2 mL vinegar),
4.61 (4 mL vinegar), 4.42 (6 mL vinegar), 4.31 (8 mL vinegar), and
4.24 (10 mL vinegar), respectively. It is therefore assumed that when
the addition level of vinegar was higher than 2 mL, the structuring effect
is significant, with a higher level of vinegar addition leading to a larger
extent of complexation (Figs. 5, 6 & 7).
By comparing the rheological properties of the prepared low-fat
mayonnaise products with the high fat mayonnaise (control), it was ob-
served that at 4–6 mL of vinegar addition level, the most similar rheo-
logical properties of the low-fat mayonnaise products could be
achieved. Subsequent sensory evaluation experiment also demon-
strated this result, showing that at 4 mL of vinegar addition, the pre-
pared product had the highest overall acceptability (Table 1). Notably,
another advantage of the fabrication strategy is that the structuring of
the low-fat mayonnaise-like products can be achieved without signifi-
cantly altering their color appearance (Fig. 4 & Table 2), because white
vinegar was used in this work to adjust the pH, and other ingredients
were unchanged. After successfully fabricating the low-fat mayonnaise
products, we evaluated their thermal stability and freeze-thaw stability
[32]. It is widely acknowledged that during heating, egg yolk protein is
unfolded leading to gelation, thus decreasing the emulsifying capacity.
As a result, the emulsion gels showed significant emulsion droplet coa-
lescence after heating treatment (Figs. 4 & 5). Moreover, freeze-thaw
treatment did not significantly affect the oil droplet morphology, indi-
cating that the emulsion gels had acceptable freeze-thaw stability.
In summary, we have shown that based on the electrostatic com-
plexation between Alg and egg yolk proteins at acidic conditions, it is
possible to design a class of emulsion gels which possess rheological
properties very similar to a full-fat mayonnaise product, but with a
lower oil content. It is also demonstrated that by altering the addition
amount of vinegar, the textural attributes of the emulsion gels can be
tunable, while maintaining the appearance color of the gels almost un-
changed. Moreover, based on the mixed Alg and egg yolk systems, we
also prepared the solid emulsions which possess acceptable
redispersibility, allowing them to be functionalized as a potential carrier
for loading functional oil soluble compounds. Overall, these results are
expected to not only deepen our understanding of food texture design
but also to widen the methodologies for fabrication of reduced-fat
foods.
Authors' statement
All authors have seen the manuscript and approved its submission.
There is no interest conflict in this manuscript either.
 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606 605

Fig. 9. Redispersibility of the lyophilized emulsion gels. (A) Visual observations; (B) optical images of the dispersions.
606 X. Yang et al. / International Journal of Biological Macromolecules 147 (2020) 595–606

Table 2
Color value determination of the prepared low-fat mayonnaise-like emulsion gels.

Sample L a b c

Fresh emulsion gels

0 mL 78.79 ± 0.4073a 0.41 ± 0.1721d 32.65 ± 0.5314bcd 32.65 ± 0.5314bcd
2 mL 77.86 ± 1.8208ab 1.12 ± 0.0907a 34.61 ± 0.9881ab 34.63 ± 0.9938ab
4 mL 78.06 ± 1.5560ab 0.87 ± 0.1709abc 31.88 ± 0.8129d 31.89 ± 0.8129d
6 mL 78.70 ± 0.1222a 1.10 ± 0.1015a 35.02 ± 0.0781a 35.04 ± 0.0814a
8 mL 78.86 ± 1.1820a 0.99 ± 0.0987ab 33.00 ± 0.8451bcd 33.02 ± 0.8508bcd
10 mL 78.79 ± 1.2125a 0.99 ± 0.1457ab 32.12 ± 1.1105d 32.14 ± 1.1161d
After freeze-thaw
0 mL 76.22 ± 1.0200bc 1.09 ± 0.0473a 33.85 ± 0.7044abcd 33.86 ± 0.7073abcd
2 mL 75.41 ± 0.9208c 1.07 ± 0.2250a 34.57 ± 0.7466abc 34.58 ± 0.7459abc
4 mL 78.53 ± 1.1333a 0.92 ± 0.1418abc 32.59 ± 0.6671cd 32.62 ± 0.6768cd
6 mL 77.68 ± 0.9614ab 1.02 ± 0.1931bcd 33.86 ± 1.5441abcd 33.89 ± 1.5471abcd
8 mL 77.95 ± 1.2033ab 0.62 ± 0.0854bc 32.14 ± 1.1400d 32.15 ± 1.1350d
10 mL 79.12 ± 0.9088a 1.08 ± 0.1900a 33.09 ± 1.9458abcd 33.10 ± 1.9511abcd

Note: Data in each column and labeled with different superscripts are significantly different (P b 0.05).

Declaration of competing interest
We declare no conflicts of interests in this work.
Acknowledgments
The study was financially sponsored by China Agriculture Research
System (CARS-28); the foundation of Key Laboratory of Urban Agricul-
ture, Ministry of Agriculture (no. UA201702), Ministry of Agriculture,
PR China; the Fundamental Research Funds for the Central Universities
(GK201603100).
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