molecules

Article
Oil-in-Water Emulsions Stabilized by Ultrasonic
Degraded Polysaccharide Complex
Yujie Li 1 , Dong Xiang 1,2, * , Bo Wang 1 and Xiaoyue Gong 1
1 College of Food Science, Hainan University, No.58 Haikou 570228, China; lyj20162012@126.com (Y.L.);
15607662867@163.com (B.W.); gxylay120408@163.com (X.G.)
2 Engineering Research Center of Utilization of Tropical Polysaccharide Resources, Ministry of Education,
No.58 Haikou 570228, China
* Correspondence: dxiang@hainu.edu.cn; Tel.: +13-976-899-293




Received: 11 February 2019; Accepted: 15 March 2019; Published: 20 March 2019


Abstract: The effects of ultrasound on the molecular weight distribution and emulsifying properties
of both xanthan gum (XG) and propylene glycol alginate (PGA) were investigated. The results showed
that ultrasonic treatment at different intensities decreased the apparent viscosity and narrowed the
molecular weight distribution. Higher intensity increased the effectivity of the sonochemical effect.
Ultrasound degradation did not change the primary structure of the PGA-XG complex, and SEM
analysis showed that the morphology of the original polysaccharide differed from that of the degraded
polysaccharide fractions. The ultrasonic intensities and treatment times had a substantial influence
on the stability of the polysaccharide-stabilized oil-in-water (O/W) emulsions. The O/W emulsion
stabilized by the polysaccharide treated with 270 W ultrasound waves for 7 min led to the smallest
average particle size (detected via fluorescence microscopy) and showed stability against aggregation
in O/W emulsions.

Keywords: ultrasound; propylene glycol alginate; xanthan gum; emulsion stability

1. Introduction
Ultrasonic waves have been applied to a variety of physical and chemical processes such as
emulsification [1], dispersion, homogenization, and various chemical reactions [2]. Furthermore,
research has mainly focused on the novel effects of ultrasound on both the properties and functionality
of products, such as the emulsification and rheological properties of food hydrocolloids [3,4].
Moreover, ultrasonic treatment is an effective and energy-saving method for preparing and processing
polymer particles [5].
Xanthan gum (XG) is a type of anionic exocellular polysaccharide produced by the aerobic
fermentation of sugars by the bacterium Xanthomonas campestris. Importantly, the rheological properties
of the aqueous phase are influenced by XG, which is used as a thickening agent and plays an essential
role in emulsion stability, contributing to the formation of a gel-like network [6]. Since XG is
a hydrophilic polymer, it preferentially enters hydrogen bonding with water. Its surface-active
properties as well as its rheological properties can be modified when it is used in combination with
propylene glycol alginate (PGA) [5].
PGA is a high molecular weight linear polysaccharide with 50–85% esterified carboxyl groups
that is derived from the reaction between propylene oxide and alginic acid [7–9] Additionally, PGA is
composed of 31–65% 1,4-linked-D-mannuronic acid and 69–35% L-guluronic acid and can be utilized
as a stabilizer and foaming agent [10,11].
The degradation of naturally occurring biopolymers has become a key goal for colloid studies, due
to the necessity of the reduction in molecular weight or particle size, or confinement within a narrow

Molecules 2019, 24, 1097; doi:10.3390/molecules24061097 www.mdpi.com/journal/molecules

Molecules
Molecules2019, 24,24,
2019, 1097 22ofof1212

narrow range size distribution [12,13], to meet application requirements [14]. Recently, several
range size distribution [12,13], to meet application requirements [14]. Recently, several techniques for
techniques for degradation have been reported, of which ultrasonic degradation was considered to
degradation have been reported, of which ultrasonic degradation was considered to be the optimum
be the optimum method for the control of the molecular weight of degraded products. In addition,
method for the control of the molecular weight of degraded products. In addition, in response to
in response to ultrasonic treatment, degraded polymers are transformed into simple chemical
ultrasonic treatment, degraded polymers are transformed into simple chemical structures, whereas
structures, whereas other methods may promote alterations and thus the behavior of materials [10].
other methods may promote alterations and thus the behavior of materials [10].
In this study, the emulsifying properties of PGA-XG complexes were characterized in response
In this study, the emulsifying properties of PGA-XG complexes were characterized in response
to treatment with various intensity levels and durations. The effects of ultrasonic treatment on the
to treatment with various intensity levels and durations. The effects of ultrasonic treatment on
molecular weight and apparent viscosity of the polysaccharide solution at two intensities were
the molecular weight and apparent viscosity of the polysaccharide solution at two intensities were
investigated as a function of time. The preliminary characterizations of the polysaccharides before
investigated as a function of time. The preliminary characterizations of the polysaccharides before
and after ultrasonic treatment were evaluated. Furthermore, the influences of ultrasonic intensity and
and after ultrasonic treatment were evaluated. Furthermore, the influences of ultrasonic intensity
treatment time on the stability of the emulsions (droplet growth and gravitational separation) were
and treatment time on the stability of the emulsions (droplet growth and gravitational separation)
determined. In addition, morphological characteristics of oil-in-water (O/W) emulsions were
were determined. In addition, morphological characteristics of oil-in-water (O/W) emulsions were
examined with fluorescence microscopy.
examined with fluorescence microscopy.

2.2.Results
Resultsand
andDiscussion
Discussion

2.1.Viscosity
2.1. ViscosityMeasurement
Measurement
Theapparent
The apparentviscosities
viscositiesofofthethePGA-XG
PGA-XGcomplexes
complexesunder underultrasonic
ultrasonictreatment
treatmentatatdifferent
different
intensitiesare
intensities areshown
shownininFigure
Figure1 as
1 as a function
a function ofof time.For
time. Forboth
bothintensities,
intensities,thetheapparent
apparentviscosity
viscosityofof
thepolysaccharide
the polysaccharidesolution
solutiondecreased
decreasedwith withtime.
time.AtAthigher
higherpower
power(390(390W),
W),thetheapparent
apparentviscosity
viscosity
decreasedsteeply
decreased steeplyfrom
fromthetheinitial
initialvalue
valueto toaaminimal
minimal level
level (similar
(similar to
to that
that of
of water)
water) within
within20 20min
minof
ofexposure
exposuretotoultrasound.
ultrasound.AtAt lower
lowerpower
power (270 W),
(270 thethe
W), apparent
apparent viscosity decreased
viscosity decreased to atominimal
a minimallevel
(similar to that of water) within 30 min of ultrasound treatment. At the beginning
level (similar to that of water) within 30 min of ultrasound treatment. At the beginning of treatment of treatment (after
10 min)
(after at 390
10 min) W,W,
at 390 thethe
apparent
apparentviscosity
viscositydecreased
decreased rapidly from8700
rapidly from 8700mPa/s
mPa/stoto300300mPa/s.
mPa/s.AsAsthethe
treatment time increased from 30 to 70 min, a decrease in the rate of the smaller
treatment time increased from 30 to 70 min, a decrease in the rate of the smaller change in apparent change in apparent
viscositywas
viscosity wasobserved
observedand andreached
reachedan an optimum
optimum value
value (15(15 mPa/s)
mPa/s)after
after7070min.
min.InIn comparison,
comparison, the
reduction
the reductionof of
apparent
apparent viscosity
viscosity was
was higher
higherandandconsiderably
considerablyfaster
fasteratathigh
highultrasonic
ultrasonicintensity.
intensity.A
stronger reduction in apparent viscosity was obtained using a higher
A stronger reduction in apparent viscosity was obtained using a higher intensity, implying that intensity, implying that
ultrasonicintensity
ultrasonic intensityhashas aa significant
significant influence
influenceon onthe
theultrasonic
ultrasonic degradation
degradation of polysaccharides.
of polysaccharides. This
degradation
This degradation maymaybe bedue
duetotocavitation
cavitationaction
action (mechanical
(mechanical effect), which increases
effect), which increaseswithwithhigher
higher
ultrasonic
ultrasonic intensity.
intensity. Subsequently,
Subsequently, this decreased
this decreased the threshold
the threshold of cavitation
of cavitation and increased
and increased the number the
ofnumber of cavitation
cavitation bubbles [15].bubbles [15].

Figure 1. Effect of ultrasonic intensities and treatment time on the apparent viscosity of PGA-XG
complexes
Figure 1.(0.06 g/L;
Effect PGA:XG 3:7
of ultrasonic wt, viscosity
intensities andmeasured 30 ◦ C).
treatmentattime on Data are expressed
the apparent as mean
viscosity ± SD
of PGA-XG
forcomplexes
three independent replicates (n = 3) of each sample.
(0.06 g/L; PGA:XG 3:7 wt, viscosity measured at 30 °C). Data are expressed as mean ± SD
for three independent replicates (n = 3) of each sample.
Molecules 2019, 24, 1097 3 of 12
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2.2.2.2.
Effect of of
Effect Ultrasound
UltrasoundononMolecular
MolecularWeight
Weight Distribution
Distribution
In In
thisthis study,
study, GPC GPC
waswas employed
employed toto investigatethe
investigate themolecular
molecularweight
weightdistribution
distributionofofthe thePGA-XG
PGA-
XG complex
complex beforebefore and ultrasonic
and after after ultrasonic treatment
treatment at different
at different intensities
intensities andand treatment
treatment times.
times. TheThe
data
data obtained are presented in Figure 2. The molecular weight distribution
obtained are presented in Figure 2. The molecular weight distribution of the PGA-XG complex of the PGA-XG complex
narrowedrapidly
narrowed rapidlywithin
withinthethefirst
first 30
30 min
min butbut slowed
slowed down
down during
duringthetheremaining
remaining30–60 30–60 min
minof of
ultrasonic
ultrasonic treatmentatat270
treatment 270 W W (Figure
(Figure 2a).2a).TheThesame
same rapid
rapidnarrowing
narrowing occurred but within
occurred the first
but within the15first
min and slowed down during the remaining 15–60 min of ultrasonic treatment
15 min and slowed down during the remaining 15–60 min of ultrasonic treatment at 390 W (Figure 2b). at 390 W (Figure 2b).
After
After ultrasonic
ultrasonic treatment,the
treatment, themolecular
molecularweightweight distribution
distribution curve
curveexhibited
exhibiteda abimodal
bimodal pattern
patternandand
did not change over time. Polysaccharide fracturing was mainly caused by the shearing force
did not change over time. Polysaccharide fracturing was mainly caused by the shearing force produced
produced by the rapid collapse of cavitation bubbles [2,16]. Large polymers with longer chains are
by the rapid collapse of cavitation bubbles [2,16]. Large polymers with longer chains are preferentially
preferentially broken by the shearing force, whereas smaller molecules are more resilient [17]. Thus,
broken by the shearing force, whereas smaller molecules are more resilient [17]. Thus, the molecular
the molecular weight distribution of the sample decreased at an early stage due to this fracturing,
weight distribution of the sample decreased at an early stage due to this fracturing, which was why
which was why the rate slowed as time progressed. The two kinds of polysaccharide were eventually
thedegraded
rate slowed as time progressed. The two kinds of polysaccharide were eventually degraded into
into different length short chains, so the molecular weight distribution curve became a
different
double peak. short chains, so the molecular weight distribution curve became a double peak.
length
Ultrasonic
Ultrasonicintensity
intensityisisananimportant
important factor affecting the
factor affecting the distinct
distinctstages
stagesofofacoustic
acoustic cavitation:
cavitation:
nucleation, bubble growth, and collapse [9]. A significant increase in degradation
nucleation, bubble growth, and collapse [9]. A significant increase in degradation efficiency was efficiency was
observed with increasing ultrasonic intensity. It can be seen from the Figure 2
observed with increasing ultrasonic intensity. It can be seen from the Figure 2 that at the same time that at the same time
point thethe
point molecular
molecularweight
weightdistribution
distributioncurvecurve of the polysaccharide
polysaccharidedegraded
degradedbyby390 390
WW is is narrower
narrower
than thethe
than molecular
molecular weight
weightdistribution
distributioncurvecurve of of the
the polysaccharide degradedby
polysaccharide degraded by270270WWultrasound.
ultrasound.

(a)

0 min
(b)

2 min 7 min

30 min 60 min
Figure 2. Cont.
Molecules 2019, 24, 1097 4 of 12
Molecules 2019, 24, 4 of 12

(c)

2 min 7 min

30 min 60 min
Figure 2. Effect
Figure Effectofof
ultrasonic treatment
ultrasonic on molecular
treatment weightweight
on molecular distribution: (a) before
distribution: (a)ultrasonic treatment,
before ultrasonic
(b) 270 W ultrasonic
treatment, treatment,treatment,
(b) 270 W ultrasonic and (c) 390and
W ultrasonic treatment.treatment.
(c) 390 W ultrasonic The red lineTherepresents the result
red line represents
of the
the light
result of scattering detector, and
the light scattering the blue
detector, and line
the represents the result the
blue line represents of aresult
differential refractive
of a differential
index detector.
refractive index detector.

2.3. Effect of Ultrasound on the Primary Structure of Polysaccharides

2.3. Effect of Ultrasound on the Primary Structure of Polysaccharides
Native and ultrasonically-treated polysaccharides, including PGA and XG (treated at ultrasonic
Native and ultrasonically-treated polysaccharides, including PGA and XG (treated at ultrasonic
intensities of 270 W and 390 W, for 60 min), were selected for further characterization of their
intensities of 270 W and 390 W, for 60 min), were selected for further characterization of their primary
primary structure.
structure.
Figure 3 shows the FTIR spectra of native and ultrasonically treated PGA and XG, all of which had
Figure 3 shows the FTIR spectra of native and ultrasonically treated PGA and XG, all of which
virtually identical characteristic absorption peaks. All samples showed a wide and strong absorption
had virtually identical characteristic absorption peaks. All samples showed a wide and strong
peak around 3429 cm−1 , which was assigned as -OH. The stretching and bending vibration absorption
absorption peak around 3429 cm−1, which was assigned as -OH. The stretching −
and bending vibration
peak at around 2924 cm−1 corresponded to C-H [18]. The peak at 1736 cm 1 could be−1 attributed
absorption peak at around 2924 cm−1 corresponded to C-H [18]. The peak at 1736 cm could be
to the stretching C=O in the acetyl groups [19]. The intense peak at 1640 cm−1 can be attributed to
attributed to the stretching C=O in the acetyl groups [19]. The intense peak at 1640 cm −1 can be
intramolecular hydrogen bonds. The strong extensive absorption peaks at 1000–1200 cm−1 suggest
attributed to intramolecular hydrogen bonds. The strong extensive absorption peaks at 1000–1200
the presence of C-O-H, C-C, and C-O-C [2,3,15], and the absorbance bands around 874 cm−1 and
cm−1 suggest the presence of C-O-H, C-C, and C-O-C [2,3,15], and the absorbance bands around 874
807 cm−1 are characteristic of mannose in PGA and XG. The high similarity in the FTIR spectra between
cm−1 and 807 cm−1 are characteristic of mannose in PGA and XG. The high similarity in the FTIR
ultrasonically-treated PGA or XG and their corresponding native forms suggest that both the repeating
spectra between ultrasonically-treated PGA or XG and their corresponding native forms suggest that
units and the primary structures of PGA and XG were not affected by the ultrasonic treatment.
both the repeating units and the primary structures of PGA and XG were not affected by the
SEM was employed as an effective tool to qualitatively analyze the changes in surface morphology
ultrasonic treatment.
of native polysaccharides (PGA and XG) and ultrasonically-treated polysaccharide fractions, and the
SEM was employed as an effective tool to qualitatively analyze the changes in surface
results are presented in Figure 4. The surfaces of the native polysaccharides and the degraded
morphology of native polysaccharides (PGA and XG) and ultrasonically-treated polysaccharide
polysaccharides showed clear variations in size and shape. The native polysaccharides (Figure 4)
fractions, and the results are presented in Figure 4. The surfaces of the native polysaccharides and
showed rough surface-like clouds or a flake-like morphology. In contrast, after ultrasonic treatment,
the degraded polysaccharides showed clear variations in size and shape. The native polysaccharides
the polysaccharide fractions (Figure 4) showed a rough appearance with extensive pores detected on
(Figure 4) showed rough surface-like clouds or a flake-like morphology. In contrast, after ultrasonic
the surface, or a mesh-like morphology. Moreover, as the ultrasonic intensity increased from 270 to
treatment, the polysaccharide fractions (Figure 4) showed a rough appearance with extensive pores
390 W, the microstructure was dramatically fragmented and small segments or pores were obtained.
detected on the surface, or a mesh-like morphology. Moreover, as the ultrasonic intensity increased
The network interconnections or aggregates were thoroughly split into thin and fragmented branches,
from 270 to 390 W, the microstructure was dramatically fragmented and small segments or pores
generated from fewer chain strands or looser structures. These results could be attributed to the
were obtained. The network interconnections or aggregates were thoroughly split into thin and
intensive cavitation, turbulent shear, and instantaneous high-pressure drop involved in the ultrasound
fragmented branches, generated from fewer chain strands or looser structures. These results could
be attributed to the intensive cavitation, turbulent shear, and instantaneous high-pressure drop
involved in the ultrasound treatment process. With increasing ultrasonic intensity and treatment
Molecules 2019, 24, 1097
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Molecules 2019, 24, 5 of 12

time, the ultrasonic energy was sufficient to break down strong bonds, such as the glycosidic linkages
time, the ultrasonic
treatment energy
process. With was sufficient
increasing to break
ultrasonic down
intensity strong
and bonds,time,
treatment suchthe
as the glycosidic
ultrasonic linkages
energy was
that connect the sugar units.
that connect
sufficient the sugar
to break down units.
strong bonds, such as the glycosidic linkages that connect the sugar units.

Figure Fourier
3. 3.
Figure Fouriertransform
transforminfrared
infrared (FTIR)
(FTIR) spectra of native
spectra of nativeand
and270
270WWand
and390
390WWultrasonically
ultrasonically
treated
treated propylene
propylene glycolalginate
glycol alginate(PGA)
(PGA)and
andxanthan
xanthan gum (XG).
(XG).
Figure 3. Fourier transform infrared (FTIR) spectra of native and 270 W and 390 W ultrasonically
treated propylene glycol alginate (PGA) and xanthan gum (XG).

(a) 0 min

(a) 0 min

(b) 270 W, 30 min (c) 390 W, 30 min

Figure
Figure 4. 4. Scanningelectron
Scanning electronmicroscopy
microscopy(SEM)
(SEM) images
images of
of polysaccharides
polysaccharidesunder
underultrasonic treatment:
ultrasonic treatment:
(a) (a) Native
Native polysaccharide;
polysaccharide; (b)
(b) 270 W, 30 min (b) 270
270 W W ultrasonic
ultrasonic treatment
treatment for 30for 30and
min; min;(c)and (c) 390 W ultrasonic
(c) 390 W, 30 min treatment
390 W ultrasonic
fortreatment
30 min. for 30 min.
Figure 4. Scanning electron microscopy (SEM) images of polysaccharides under ultrasonic treatment:
2.4.2.4.
Effect
(a) of of
Effect Ultrasound
Native Ultrasound ononInterfacial
polysaccharide;Interfacial
(b) 270Tension
Tension
W ultrasonic treatment for 30 min; and (c) 390 W ultrasonic
treatment for 30 min.
The interfacial
The interfacialcharacteristics
characteristicsofofsurface-active
surface-active ingredients areaasignificant
ingredients are significantfactor
factorinin determining
determining
their
their ability
ability toto form
form andstabilize
and stabilizeemulsions
emulsions[16].[16]. This study
studyexamined
examinedthetheinterfacial
interfacialtension
tensionbetween
between
2.4. Effect of Ultrasound on Interfacial Tension
polysaccharide
polysaccharide solutions
solutions andand coconut
coconut oil (Figure
oil (Figure 5). The 5).experimental
The experimental results indicated
results indicated that
that ultrasonic
ultrasonic
power The
reduces power
interfacial reduces tension
the interfacial the interfacial
characteristics tension ingredients
ofatsurface-active
the oil-water at the oil-water
interface, a interface,
areindicating
thus significant thus indicating
factor
that that
in determining
ultrasonic treatment
theirchange
can ability to
theform and stabilize
adsorption emulsions [16].
characteristics of theThis study examined
PGA-XG complexes theatinterfacial tension
this interface between
(Figure 5).
polysaccharide solutions and coconut oil (Figure 5). The experimental results indicated that
ultrasonic power reduces the interfacial tension at the oil-water interface, thus indicating that
Molecules 2019, 24, 6 of 12
Molecules 2019, 24, 1097 6 of 12
ultrasonic treatment can change the adsorption characteristics of the PGA-XG complexes at this
interface (Figure 5). However, different ultrasonic intensities and treatment times showed variations
However, different ultrasonic intensities and treatment times showed variations in their ability to
in their ability to decrease interfacial tension at the O/W interface. We found that PGA-XG complexes
decrease interfacial tension at the O/W interface. We found that PGA-XG complexes significantly
significantly reduced the interfacial tension compared to water (23.5 mN/m) at coconut oil–
reduced the interfacial tension compared to water (23.5 mN/m) at coconut oil–polysaccharide solution
polysaccharide solution interfaces. The presence of both hydrophilic and hydrophobic bonds enabled
interfaces. The presence of both hydrophilic and hydrophobic bonds enabled the effective adsorption
the effective adsorption of the PGA-XG mixture at the interface of the coconut oil. According to Figure
of the PGA-XG mixture at the interface of the coconut oil. According to Figure 5, during the early stages
5, during the early stages of ultrasonic treatment, the interfacial tension between the oil and
of ultrasonic treatment, the interfacial tension between the oil and polysaccharide solution decreased
polysaccharide solution decreased rapidly, which was consistent with the results of apparent
rapidly, which was consistent with the results of apparent viscosity. Furthermore, the amphiphilic
viscosity. Furthermore, the amphiphilic polysaccharides generated thick interfacial layers after they
polysaccharides generated thick interfacial layers after they were adsorbed onto the oil droplet surface.
were adsorbed onto the oil droplet surface. The cavitation of the ultrasonic power degrades
The cavitation of the ultrasonic power degrades polysaccharides into smaller fractions [4,20], which can
polysaccharides into smaller fractions [4,20], which can be adsorbed quicker to the oil droplet surface,
be adsorbed
where they form quicker
a layerto[21],
the which
oil droplet
maysurface, where
contribute they
to the form a of
decrease layer [21], which
interfacial may contribute to
tension.
the decrease of interfacial tension.

Figure 5. Effect of different ultrasonic intensities and treatment times on the interfacial tension
Figure 5. Effect
measured of coconut
at the differentoil–polysaccharide
ultrasonic intensities and interface.
solution treatmentData
timesareonexpressed
the interfacial
as meantension
± SD of
measured at the coconut
three independent oil–polysaccharide
replicates (n = 3) of eachsolution
sample.interface. Data are expressed as mean ± SD of
three independent replicates (n = 3) of each sample.
2.5. Particle Size and Size Distribution of Fresh Emulsions
2.5. Particle Size and
Ultrasonic Size Distribution
intensity of Fresh
and treatment Emulsions
times can greatly influence O/W emulsion stability; therefore,
appropriate
Ultrasonicparameters
intensity and must be foundtimes
treatment to optimize
can greatlythe shelf life ofO/W
influence emulsions.
emulsion The averagetherefore,
stability; droplet size
and distributions
appropriate parametersof 5% coconut
must oil O/W
be found emulsions
to optimize thestabilized
shelf life by PGA-XG complexes
of emulsions. The average anddroplet
prepared
by different ultrasonic intensities and treatment times were tested.
size and distributions of 5% coconut oil O/W emulsions stabilized by PGA-XG complexes and The droplet size distribution
and average
prepared particle ultrasonic
by different size of coconut oil O/W
intensities andemulsions
treatmentare shown
times were in tested.
Figures The
6 and 7. Thesize
droplet shear
rate of ultrasonic
distribution and averagepower decreased
particle size ofthe droplet
coconut size of
oil O/W the coconut
emulsions oil in in
are shown theFigure
O/W6emulsion
and Figure [22].
7. The shear rate of ultrasonic power decreased the droplet size of the coconut oil in the O/W emulsionthe
The mixture of PGA and XG formed a film around the droplet surface, which better maintained
stability
[22]. of the of
The mixture 5%PGAcoconut
and XG oil O/W
formed emulsions
a film around[23]. the
Thedroplet
polysaccharides formed
surface, which a relatively
better maintainedthick
hydrophilic coating around the coconut oil droplets and generated a strong
the stability of the 5% coconut oil O/W emulsions [23]. The polysaccharides formed a relatively thick steric repulsion that
hydrophilic coating around the coconut oil droplets and generated a strong steric repulsion thatthe
prevented aggregation in the O/W emulsions. As shown in Figure 6, the average particle size of
O/W emulsions
prevented aggregationprepared
in theby O/Wa 270 W intensity
emulsions. As ultrasound
shown in Figurewave 6, wasthesignificantly smaller
average particle than
size those
of the
O/W emulsions prepared by a 270 W intensity ultrasound wave was significantly smaller than thosethe
prepared by 390 W. The experimental results showed that the ultrasonic time greatly influenced
averageby
prepared particle
390 W.sizeTheofexperimental
the emulsions. The O/W
results showed emulsion
that theprepared
ultrasonicby time
270 W for 7 min
greatly exhibited
influenced thethe
smallest
average average
particle sizeparticle size.
of the emulsions. The O/W emulsion prepared by 270 W for 7 min exhibited the
smallest average particle size.the emulsions prepared by 270 W ultrasound for 7 min and 2 min presented
As shown in Figure 7,
theAsnarrowest
shown indroplet
Figuresize distribution
7, the emulsions and resultedby
prepared in 270
the best stability. However,
W ultrasound for 7 min theand
droplet
2 min size
presented the narrowest droplet size distribution and resulted in the best stability. However, thethe
distribution of the emulsions widened after prolonged ultrasonic treatment. This may be related to
reduction
droplet in emulsion of
size distribution viscosity, causingwidened
the emulsions partial aggregation.
after prolonged However, all droplet
ultrasonic size distributions
treatment. This may be of
the coconut
related oil O/W emulsions
to the reduction in emulsionprepared
viscosity,bycausing
390 W ultrasonic waves were
partial aggregation. wider than
However, that prepared
all droplet size
Molecules 2019, 24, 7 of 12
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Molecules 2019, 24, 1097 7 of 12
distributions of the coconut oil O/W emulsions prepared by 390 W ultrasonic waves were wider than
distributions
that prepared by of the
270coconut oil O/W
W ultrasonic emulsions
waves. prepared
This may by 390 the
be because W ultrasonic wavesultrasonic
high-intensity were wider than
effect
that
can prepared
by degrade by 270 W ultrasonic
the polysaccharide
270 W ultrasonic waves. Thischainwaves.
mayinto This may
shorter the
be because be
chains; because the
therefore, the
high-intensity high-intensity
effect ofeffect
ultrasonic ultrasonic effect
the polysaccharide
can degrade the
can degrade
chain the
in stabilizing
polysaccharide polysaccharide
theinto
chain O/Wshorter chain
emulsions isinto
chains; shorterthe
worse.
therefore, chains;
effecttherefore, the effect of chain
of the polysaccharide the polysaccharide
in stabilizing
chain in stabilizing the
the O/W emulsions is worse. O/W emulsions is worse.

Figure 6. Average droplet size of 5% coconut oil O/W emulsions prepared by different ultrasonic
Figure
intensity 6. Average
Figure 6.
and Average droplet
droplet
treatment
size of
times.size
Data
5%
ofare coconut
coconutoil
5%expressed oilO/W
O/W emulsions
as mean emulsions prepared
prepared
± SD for three
by
by different
different
independent
ultrasonic
ultrasonic
replicates (n =
intensity
intensity and
and treatment
treatmenttimes.
times.Data
Dataare expressed
are as
expressed mean
as ±
mean SD
± for
SD three
for independent
three independentreplicates (n =(n3)=
replicates
3) of each sample.
of
3) each sample.
of each sample.

(a) (b)
(a) (b)
Figure
Figure7.7.Droplet
Dropletsize
sizedistribution
distributionofof5% 5%coconut
coconutoiloilO/W
O/W emulsions
emulsionsstabilized byby
stabilized different
differentultrasonic
ultrasonic
Figure 7.and
intensities
intensities Droplet size distribution
andtreatment
treatment times: ofDroplet
5% coconut
times:(a)(a)Droplet oil O/W emulsions
sizedistribution
size distribution ofofO/W
O/W stabilized
emulsions
emulsions by differentbyultrasonic
prepared
prepared by270
270WW
intensities
ultrasonic
ultrasonic and treatment
power
power and(b)
and times:size
(b)droplet
droplet (a) Droplet sizeofdistribution
sizedistribution
distribution ofO/W
O/W of O/W
emulsions
emulsions emulsions
prepared
prepared byby prepared
390W.
390 W. by 270 W
ultrasonic power and (b) droplet size distribution of O/W emulsions prepared by 390 W.
2.6.Microstructure
2.6. Microstructureofofthe theEmulsions
Emulsions
2.6. Microstructure
Thefluorescence
fluorescence of the Emulsions images of the 5% coconut O/W emulsions stabilized by PGA-XG
microscopic
The microscopic images of the 5% coconut O/W emulsions stabilized by PGA-XG
complexes
The prepared
fluorescence by different
microscopic
complexes prepared by different ultrasonic ultrasonic intensities
imagesintensities
of the 5% and andtreatment
treatment
coconut timesare
O/W emulsions
times arepresented
presentedinby
stabilized in Figure
PGA-XG
Figure 8.8.
Compared to the image of pre-emulsion (Figure 8a), the droplet sizes of PGA-XG
Compared to the image of pre-emulsion (Figure 8a), the droplet sizes of PGA-XG (3:7, wt) stabilized8.
complexes prepared by different ultrasonic intensities and treatment times are (3:7,
presentedwt) stabilized
in Figure
emulsions
Compared
emulsions were
to the
were slightly
slightly smaller
imagesmaller afterultrasonic
of pre-emulsion
after ultrasonic
(Figure treatment andshowed
8a), the droplet
treatment and showed
sizes of insignificant
PGA-XG (3:7,
insignificant variation when
wt) stabilized
variation when
the
the ultrasonic
emulsions
ultrasonic were exposure
slightly
exposure was
was extended.
smaller Likely,the
afterLikely,
extended. ultrasonic thetreatment
ultrasonicand
ultrasonic waves
showed
waves decreased
decreased thedroplet
droplet
insignificant
the sizeofof
variation
size the
when
the
internal
the phase
ultrasonic due to
exposure cavitation
was [24].
extended. However,
Likely, the with prolonged
ultrasonic waves time the
decreased
internal phase due to cavitation [24]. However, with prolonged time the phenomenon of droplet phenomenon
the droplet of droplet
size of the
aggregation
internal phase
aggregation occurred
due to
occurred in the O/W
in cavitation emulsions, which
[24]. However,
the O/W emulsions, whichwithmay be related
may prolonged to
be related to the
time low
thethe apparent
lowphenomenon viscosity of the
of droplet
apparent viscosity of
the O/W emulsions (Figure 8c). As shown in Figure 8b, the O/W emulsions prepared by intensity
O/W emulsions
aggregation (Figure
occurred in 8c).
the As
O/Wshown in Figure
emulsions, 8b,
which the
may O/W
be emulsions
related to prepared
the low by 270
apparent Wviscosity
270 Wof
for
the7O/W
intensitymin and
for 15 min
emulsions
7 min presented
and(Figure a small
8c).
15 min presentedAs showndroplet
a smallinsize.
Figure size.
droplet 8b, the O/W emulsions prepared by 270 W
intensity for 7 min and 15 min presented a small droplet size.
Molecules 2019, 24, 1097 8 of 12
Molecules 2019, 24, 8 of 12

(a)

Pre-emulsion
(b)

2 min 7 min 15 min

30 min 60 min
(c)

2 min 7 min 15 min

30 min 60 min
Figure8.8.Microstructures
Figure Microstructures of
of fresh coconut
coconut(O/W)
(O/W)emulsions
emulsions treated byby
treated ultrasonic waves
ultrasonic andand
waves observed
observed
viafluorescence
via fluorescencemicroscopy:
microscopy: (a) pretreatment
pretreatmentemulsion,
emulsion,(b)
(b)O/W
O/W emulsions
emulsions treated by 270
treated W, and
by 270 (c) (c)
W, and
O/W emulsions treated by 390
O/W emulsions treated by 390 W. W.

2.7. Visual Phase Separation

Molecules 2019, 24, 1097 9 of 12

Molecules 2019, 24, 9 of 12

2.7. Visual Phase Separation
appearance of of the
the emulsions
emulsions after
after storage
storage at
at 30 ◦ C for a period of time is shown in Figure 9.
30 °C
The appearance for a period of time is shown in Figure 9.
After 30 days, partial phase separation was observed.observed. The O/W emulsions prepared
O/W emulsions prepared by
by 270
270 WW for
for
both 22 and
both and77minminwerewerestable
stableagainst
againstgravitation
gravitation separation.
separation. TheThe majority
majority of those
of those prepared
prepared by
by 390
390showed
W W showed phasephase separation,
separation, whichwhich is consistent
is consistent with
with the the results
results of the average
of the average droplet droplet size
size (Figure
(Figure
9, after 9,30after
days30 days of storage).
of storage). High-intensity
High-intensity andand long-term
long-term ultrasonictreatment
ultrasonic treatmentdegraded
degraded the
polysaccharide into
polysaccharide into short chains, which have a poor ability
ability to
to stabilize
stabilize O/W emulsions. Therefore,
O/W emulsions. Therefore,
phase separation occurred in the O/W emulsionsduring
O/W emulsions duringstorage.
storage.

2 min 7 min 15 min 30 min 60 min 2 min 7 min 15 min 30 min 60 min

(a) (b)
Figure
Figure 9. General
General appearance
appearance of
of 5% coconut oil O/W
O/W emulsions
emulsions prepared
prepared by
by different ultrasonic
intensities and treatment times after 30 days of storage at 30 ◦°C:
intensities C: (a) 270 W and (b) 390 W.

3. Materials
3. Materials and
and Methods
Methods

3.1. Materials and Polysaccharide Solution Preparation

3.1. Materials and Polysaccharide Solution Preparation
PGA (75% degree of esterification) was purchased from Yuanye Bio-Technology Co., Ltd.
PGA (75% degree of esterification) was purchased from Yuanye Bio-Technology Co., Ltd.
(Shanghai, China). Xanthan gum was purchased from Sigma-Aldrich (St. Louis, MO, USA),
(Shanghai, China). Xanthan gum was purchased from Sigma-Aldrich (St. Louis, MO, USA), and
and commercial coconut oil was purchased from a local supermarket (QianCheng food company,
commercial coconut oil was purchased from a local supermarket (QianCheng food company, Hainan,
Hainan, China). Nile Red was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China).
China). Nile Red was purchased from Yuanye Bio-Technology Co., Ltd. (Shanghai, China). All other
All other chemicals utilized were of analytical grade and were purchased from Sinopharm Chemical
chemicals utilized were of analytical grade and were purchased from Sinopharm Chemical Reagent
Reagent Co., Ltd. (Shanghai, China). Purified water was prepared using a Barnstead E-pure system
Co., Ltd. (Shanghai, China). Purified water was prepared using a Barnstead E-pure system (Dubuque,
(Dubuque, IA, USA).
IA, USA).
The polysaccharide solution was prepared as follows: 0.18 g of PGA and 0.42 g XG were dispersed
The polysaccharide solution was prepared as follows: 0.18 g of PGA and 0.42 g XG were
in 100 mL pure water and gently stirred at 30 ◦ C. The dispersion was incubated overnight to ensure
dispersed in 100 mL pure water and gently stirred at 30 °C. The dispersion was incubated overnight
complete hydration of the complex.
to ensure complete hydration of the complex.
3.2. Viscosity Measurement
3.2. Viscosity Measurement
Apparent viscosity measurements were carried out using a Brookfield viscometer (DV3TLVTJ0,
Apparent viscosity measurements were carried out using a Brookfield viscometer (DV3TLVTJ0,
Middleboro, MA, USA) equipped with a spindle numbered LV-1 or LV-2 at 50 r/min. The sample cell
Middleboro, MA, USA) equipped with a spindle numbered LV-1 or LV-2 at 50 r/min.◦ The sample cell
was filled with the polysaccharide solution. The temperature was maintained at 30 C by circulating
was filled with the polysaccharide solution. The temperature was maintained at 30 °C by circulating
water from a constant temperature circulator.
water from a constant temperature circulator.
3.3. Preparation of O/W Emulsions
3.3. Preparation of O/W Emulsions
Coconut oil (5 g) was added to the 95 g as-prepared polysaccharide solution (0.6% wt). The mixture
Coconut
was then oil (5 g) was added
pre-homogenized to theshear
with a high 95 ghomogenizer
as-prepared polysaccharide
(FJ-200; Biaobensolution (0.6%Shanghai,
Instruments, wt). The
mixture was then pre-homogenized with a high◦ shear homogenizer (FJ-200; Biaoben
China) at a speed of 18,000 rpm for 2 min at 30 C. The resulting coarse emulsion was treated with Instruments,
Shanghai,
ultrasoundChina)
wavesat(FS-600N,
a speed of 18,000 rpm
Shangchao, for 2 min
Shanghai, at 30 at
China) °C.a The resulting
constant power coarse
of 270emulsion
W or 390was
W
treated with ultrasound waves (FS-600N, Shangchao, Shanghai, China) at a constant power
for 2 min, 7 min, 15 min, 30 min, and 60 min. The coarse emulsion was placed into a 200 mL glass of 270 W
or 390 W forcold
containing 2 min, 7 min,
water 15 min, overheating
to prevent 30 min, and 60of min. The coarse
the emulsion emulsion
during was placed
ultrasound into a 200
treatment. mL
Finally,
glass
0.005 containing cold (NaN
g sodium azide water )to prevent overheating of the emulsion during ultrasound treatment.
3 was added as an antimicrobial preservative.
Finally, 0.005 g sodium azide (NaN3) was added as an antimicrobial preservative.

3.4. Determination of Average Molecular Weight and Molecular Weight Distribution

Molecules 2019, 24, 1097 10 of 12

3.4. Determination of Average Molecular Weight and Molecular Weight Distribution

The molecular weight distribution was determined by gel permeation chromatography (GPC)
following the method described by Houben [25] with minor modifications. 20 µL of the sample solution
was injected and eluted with 0.02% NaN3 at 40 ◦ C at a flow rate of 0.5 mL/min. The eluent was
monitored with both a Waters 515 laser light scattering detector (Water, MA, USA) and a differential
refractive index detector (Water, MA, USA).

3.5. Fourier Transform Infrared (FTIR) Spectroscopic Analysis

The primary structure of the freeze-dried PGA-XG complexes before and after ultrasonic treatment
was investigated using a FTIR spectrometer (Bruker Optics Co., Berlin, Germany) at a wavenumber
range of 500–4000 cm−1 using potassium bromide (KBr) pellets.

3.6. Interfacial Characteristics Measurement

The interfacial tension between the oil and water was measured by a drop-shape analysis
instrument (DropMeter A-60, MAIST, Ningbo, China) at 30 ◦ C, according to a previously reported
method [11]. The screw control above the injector was twisted to form a suitable oil droplet size and to
measure the falling droplet. The droplets were allowed to stand in the coconut oil for 5 min.

3.7. Scanning Electron Microscopy (SEM)

The freeze-dried polysaccharide samples were fixed onto a copper stub. After sputtering with
a layer of gold, SEM images were observed and recorded using a SEM (Cannon Co., Tokyo, Japan)
under high vacuum conditions at accelerating voltage.

3.8. Measurement of Droplet Size

The average droplet size and distribution of coconut oil O/W emulsions were determined by
a laser particle size analyzer (Ineas physical optics instrument, Co., Ltd., WJ-60, Shanghai, China).
To measure both the average particle size and particle size distribution, 0.5 mL of the sample was
added to the sample cell. The experiment was conducted at 30 ◦ C. The average particle size and the
particle size distribution curve were obtained with the software WJ-60 that came with the instrument.

3.9. Fluorescence Microscopy

A fluorescence microscope (Leica DMI DM6000B, Leica Microsystems, Heidelberg, Germany) was
used to observe the microstructure of the O/W emulsions. This microscope operated in fluorescence
mode using a 10× objective with numerical aperture of 0.40. The oil droplets were stained with Nile
Red fluorescent dye. An aliquot of emulsion sample was placed onto a microscope slide. A cover
slip was placed on top of the microscope slide, ensuring that no air bubbles were trapped inside.
The samples were observed at 30 ◦ C.

3.10. Visual Observation of the Phase Separation

The O/W emulsions (10 mL) were transferred to transparent glass test tubes, sealed with plastic
caps, and stored at 30 ◦ C for 30 days. Physical phase separation was monitored.

3.11. Statistical Analysis

All measurements were performed in triplicate, and statistical analysis was performed using the
Statistical Analysis Systems (Origin 9.0, Originlab, Studio, CA, USA) software package.
Molecules 2019, 24, 1097 11 of 12

4. Conclusions
In conclusion, the primary characterization, molecular weight, and effect of ultrasonic treatment
on the stability of O/W emulsions of PGA-XG complexes were investigated. The results obtained
show that the complexes had a large reduction in apparent viscosity. The preliminary structure of the
PGA-XG complex before and after ultrasonic treatment did not change. According to SEM analysis,
the morphology of the original complexes differed from the degraded polysaccharide fractions and
exhibited variations in the maintenance of the stability of the O/W emulsions. At a specific ultrasonic
power, small and stable oil droplets formed in the polysaccharide mixture, which prevented droplet
aggregation due to the formation of a dense film at the interface. Different ultrasonic intensities and
treatment times affected the degradation of polysaccharides. The O/W emulsion treated at 270 W
for 7 min exhibited the best stability. This may be related to the molecular weight distribution of
polysaccharide fractions, which is uniform under high-power long-term treatment, and the apparent
viscosities of the O/W emulsions being too low to maintain stability. The O/W emulsion subjected to
short-time, low-power treatment achieved high apparent viscosity and resisted aggregation. However,
treatment with low ultrasonic power was insufficient to break the oil droplets into particles with
a small diameter. Therefore, ultrasonic power is a useful means for the degradation of polysaccharide
and O/W emulsion preparations. In subsequent studies, the effect of ultrasonic treatment on the
rheological properties of PGA and XG mixtures will be further studied.

Author Contributions: Conceptualization, D.X.; Methodology, D.X. and Y.L.; Software, X.G..; Validation, Y.L.,
B.W. and X.G..; Formal Analysis, Y.L.; Investigation, Y.L., B.W. And X.G.; Resources, D.X.; Data Curation, Y.L.;
Writing-Original Draft Preparation, Y.L., X.G. and B.W.; Writing-Review & Editing, D.X.; Visualization, Y.L.;
Supervision, D.X.; Project Administration, D.X.; Funding Acquisition, D.X.
Funding: The authors gratefully acknowledge the financial support received from the Provincial Key Research
and Development Project, Hainan province, China (ZDYF2018071).
Conflicts of Interest: The authors declare no conflict of interest.

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Sample Availability: Samples of the compounds are not available from the authors.

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