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\begin{document}

\title{Stabilization of liquid water-in-oil emulsions by modifying the
interfacial interaction of glycerol monooleate with aqueous phase
ingredients}



\author[1]{Maria Romero-Peña}%
\author[1]{Supratim Ghosh}%
\affil[1]{University of Saskatchewan}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Glycerol monooleate (GMO)-stabilized liquid water-in-vegetable oil
(W/VO) emulsions are difficult to stabilize due to the desorption of GMO
from the W-VO interface towards the oil phase. This work improved the
stability of GMO-stabilized liquid 20 wt\% water-in-canola oil (W/CO)
emulsion by modifying the dispersed aqueous phase composition with
hydrogen bond-forming agents. As a control, 20 wt\% water-in-mineral oil
(W/MO) emulsion was also utilized. Different concentrations of hydrogen
bond-forming agents (citric acid (CA), ascorbic acid (AA), low methoxyl
pectin (LMP)) with and without salts (sodium chloride (S) or calcium
chloride (Ca)) was added to the aqueous phase before emulsification,
which enhanced emulsifier binding to the water-oil interface. The
emulsions were characterized by phase separation, stability against
accelerated gravitation, microstructure and rheology. W/CO emulsion
without any aqueous phase additive destabilized instantly, whereas W/MO
emulsion stayed stable. The addition of hydrogen bond-forming agents and
salts significantly improved emulsion stability. LMP, with many hydrogen
bond-forming groups, was able to provide the highest emulsion stability
after 7 days in both oils compared to AA, CA and their mixtures with S.
Emulsions with both oils formed weak gels with viscous and elastic
characteristics due to the formation of an extensive network of water
droplet aggregates. Overall, the hydrogen bond-forming agents interacted
with GMO at the interface, thereby improving their presence at the water
droplet surface, allowing significantly improved stability of
GMO-stabilized liquid W/CO emulsions. The knowledge developed in this
research can be useful in applying GMO in stabilizing liquid
water-in-oil emulsion without using any crystal network.%
\end{abstract}%



\sloppy


\textbf{Stabilization of liquid water-in-oil emulsions by modifying the
interfacial interaction ofglycerol monooleate with aqueous phase
ingredients}

Maria Romero-Pe\selectlanguage{ngerman}ña\textsuperscript{a,b}, Supratim
Ghosh\textsuperscript{a*}

\textsuperscript{a}\emph{Department of Food and Bioproduct Sciences,
College of Agriculture and Bioresources, University of Saskatchewan,
Saskatoon, Saskatchewan S7N 5A8, Canada}

\textsuperscript{b}\emph{Escuela Superior Politécnica del Litoral,
ESPOL, Facultad de Ingeniería en Mecánica y Ciencias de la Producción,
Campus Gustavo Galindo Km. 30.5 Vía Perimetral, P.O. Box 09-01-5863,
Guayaquil, Ecuador}

*Corresponding authors contact

E-mail: supratim.ghosh@usask.ca

\textbf{ABSTRACT}

Glycerol monooleate (GMO)-stabilized liquid water-in-vegetable oil
(W/VO) emulsions are difficult to stabilize due to the desorption of GMO
from the W-VO interface towards the oil phase. This work improved the
stability of GMO-stabilized liquid 20 wt\% water-in-canola oil (W/CO)
emulsion by modifying the dispersed aqueous phase composition with
hydrogen bond-forming agents. As a control, 20 wt\% water-in-mineral oil
(W/MO) emulsion was also utilized. Different concentrations of hydrogen
bond-forming agents (citric acid (CA), ascorbic acid (AA), low methoxyl
pectin (LMP)) with and without salts (sodium chloride (S) or calcium
chloride (Ca)) was added to the aqueous phase before emulsification,
which enhanced emulsifier binding to the water-oil interface. The
emulsions were characterized by phase separation, stability against
accelerated gravitation, microstructure and rheology. W/CO emulsion
without any aqueous phase additive destabilized instantly, whereas W/MO
emulsion stayed stable. The addition of hydrogen bond-forming agents and
salts significantly improved emulsion stability. LMP, with many hydrogen
bond-forming groups, was able to provide the highest emulsion stability
after 7 days in both oils compared to AA, CA and their mixtures with S.
Emulsions with both oils formed weak gels with viscous and elastic
characteristics due to the formation of an extensive network of water
droplet aggregates. Overall, the hydrogen bond-forming agents interacted
with GMO at the interface, thereby improving their presence at the water
droplet surface, allowing significantly improved stability of
GMO-stabilized liquid W/CO emulsions. The knowledge developed in this
research can be useful in applying GMO in stabilizing liquid
water-in-oil emulsion without using any crystal network.

\textbf{Keywords:} water-in-oil emulsion; glycerol monooleate; hydrogen
bond-forming agents; emulsion stability; interfacial interaction

\textbf{1. Introduction}

Products containing water-in-oil (W/O) emulsions are widely used in the
pharmaceutical, cosmetic, petroleum and food industries. In food, almost
all water-in-vegetable oil (W/VO) emulsions (e.g., margarine) are used
in a semi-solid state (Ushikubo \& Cunha, 2014). The emulsion
stabilization mechanism in margarin is mainly due to fat crystal-based
Pickering and network formation (Ghosh \& Rousseau, 2011). Emulsifiers
are also critical in the formation of W/O emulsion. Most emulsifiers
used to form W/O emulsions are non-ionic lipophilic molecules (e.g.,
polyglycerol polyricinoleate (PGPR), Spans, monoglycerides). The
selection of emulsifiers is based on their hydrophilic-lipophilic
balance (HLB), which must be low (3 -- 6) for W/O emulsion. Various
research groups have worked with different combinations of oil,
emulsifiers and aqueous phase ingredients to obtain stable liquid W/O
emulsions. Opawale and Burgess (1998) investigated concentrated 50 wt\%
water-in-light mineral oil emulsions with Span 20, 80, 83 and 85 (1 -- 5
\%w/v) in the oil and sodium chloride (0 -- 1 M) in the aqueous phase.
The addition of the salt (S) within an optimum range enhanced the
stability of the emulsion. Scherze et al. (2006) investigated
stabilization of 30 wt\% water-in-medium-chain triglyceride (MCT) oil
emulsion with PGPR (4 wt\%) and soy lecithin (2.5 wt\%) in the presence
of 0.6 \% S in the aqueous phase. For lecithin, S favoured water droplet
coalescence; however, for PGPR, the incorporation of S was necessary to
obtain a stable W/O emulsion (Scherze et al., 2006). Lindenstruth and
Muller (2004) investigated 30 wt\% W/VO emulsions prepared with a 1:1
mixture of olive oil and MCT with 20\% soya lecithin as an emulsifier.
It was proposed that the \emph{cis} double bond of oleic acid needed
more space at the W-O interface, which prevented efficient interfacial
packing of lecithin (Lindenstruth \& Muller, 2004). In another research,
Márquez et al. (2010) prepared 20 -- 40 wt\% W/VO emulsion using
sunflower oil with 0.2 -- 1 wt\% PGPR and an aqueous phase containing
various concentrations of calcium chloride and other salts (calcium
lactate or carbonate; sodium, magnesium or potassium chloride). The
incorporation of any salt allowed emulsion stabilization with higher
water content. The stabilization effect of calcium salts was associated
with a reduced attractive force between the water droplets and a higher
PGPR adsorption density at the droplet surface. Prichapan et al. (2017)
prepared 20 wt\% W/VO emulsions with rice bran oil and stabilized them
with rice bran stearin and PGPR. Emulsions without rice bran stearin (a
source of fat crystals) were highly unstable to phase separation after
one day; however, the stability improved by increasing the PGPR
concentration from 2 to 4 wt\% due to the reduction in water droplet
size. However, the most stable emulsion was obtained with an oil phase
containing 45 wt\% rice bran stearin, which indicates that incorporating
fat crystals as a stabilizing factor was more important than increasing
the PGPR concentration.

PGPR is one of the most commonly used emulsifiers to develop food-grade
W/O emulsions for its ability to stabilize water droplets by a polymeric
steric barrier (Goubran \& Garti, 1988; Mettu et al., 2018). Although it
is classified as GRAS (generally recognized as safe), in 1979, the
Scientific Committee for Food of the European Community determined an
acceptable daily intake of 7.5 mg/kg body weight. In 1980, the Canadian
Regulations reduced the maximum concentration of PGPR to 0.25\% in
chocolate (Wilson et al., 1998). However, in 2017, PGPR was limited just
for chocolate products with a maximum of 0.5 wt\% (Canada, 2017). For
this reason, different groups investigated various combinations of PGPR
with other ingredients to lower its concentration in W/VO emulsion
(Ghosh \& Rousseau, 2009; Rafanan \& Rousseau, 2017). Nevertheless,
there is an urgent need to completely replace PGPR from W/VO emulsions
with a more natural small molecule emulsifier. Hence, one of the aims of
the present study was to develop stable liquid food-grade W/VO emulsions
by completely replacing PGPR with glycerol monooleate (GMO) without any
stabilizing fat crystals. GMO is a widely studied low HLB (3 -- 4)
emulsifier that is non-toxic, biodegradable, and classified as GRAS. It
is commonly used to prepare margarine-type semi-solid emulsions;
however, the presence of saturated fat crystals is essential for
emulsion stability (Ghosh \& Rousseau, 2011). To our knowledge, no
research so far has investigated stabilizing liquid W/VO emulsions
without using PGPR and saturated fat crystals. The challenge here is
that liquid W/VO emulsions prepared solely with GMO are impossible to
stabilize due to its desorption from the water droplet interface towards
VO. Ghosh et al. (2011) observed that GMO's desorption from the W-VO
interface strongly relies on its stronger hydrogen bonding interactions
with the VO triacylglycerol (TAG) fatty acid carboxylic groups than the
weaker hydrogen bonding interaction with water hydroxyl groups. The
authors also showed that, in the presence of a hydrocarbon oil (such as
mineral oil), GMO could very well remain at the water droplet surface
due to the lack of interaction with the oil, thereby improving emulsion
stability (Ghosh et al., 2011). Therefore, we hypothesized that the
presence of hydrogen bond-forming molecules in the dispersed aqueous
phase would form strong hydrogen bonds with GMO at the W-O interface,
thereby improving the stability of liquid W/VO emulsions. Three
different hydrogen bond-forming agents (two small molecules with
multiple carboxylic groups, ascorbic acid (AA), and citric acid (CA) and
one biopolymer, low methoxyl pectin (LMP)) with or without the presence
of salts (sodium chloride (S) or calcium chloride (Ca)) were
investigated to improve the stability of water-in-canola oil (W/CO)
emulsions. As a control, light mineral oil (MO, made of paraffin
hydrocarbons) was also used to form water-in-mineral oil (W/MO)
emulsions, and its influence on emulsion stability and rheology was
compared to W/CO emulsions.

\par\null

\textbf{2. Materials and methods}

2.1 Materials

Canola oil (CO) was purchased from a local grocery store and stored at 4
°C. Distilled monoglycerides DMG 0298 (90 -- 95\% monoglycerides,
containing mainly 75 -- 91\% oleic, 2 -- 17\% linoleic, 3 -- 6\%
stearic, and 3 -- 5\% palmitic) was donated by Palsgaard (Juelsminde,
Denmark). L-ascorbic acid (AA) and Nile red were purchased from Sigma
Aldrich (Oakville, ON, Canada). Citric acid anhydrous (CA), light
mineral oil (MO, code O121-4), and hydrochloric acid (HCl) (1 N) were
purchased from Fisher Scientific (Toronto, ON, Canada). Sodium chloride
(S) and calcium chloride (Ca) were obtained from BDH (VWR International,
USA). Low methoxyl pectin (LMP) amidated, Genu\textsuperscript{®} pectin
type LM-101 (degree of esterification 36\% and degree of amidation 14\%)
was provided by CP Kelco (Lille Skensved, Denmark). Purified water by
Milli-Q\textsuperscript{TM} (Millipore Corporation, MA, USA) was used
for the dispersed phase.

2.2 Preparation of solutions

AA and CA aqueous solutions were prepared with 0.125 and 0.5 wt\% acids
with 0 and 0.125, wt\% S at room temperature with stirring (400 rpm) and
stored at 4 °C. LMP solutions at pH 3.0 with 1N HCl were prepared with
1.5 and 2 wt\% LMP with 0 and 0.045 wt\% Ca at 90 ± 0.5 °C, stirred at
200 rpm for 20 min. The final LMP solutions were cooled down to room
temperature before W/O emulsions preparation. The concentrations of the
ingredients were selected based on preliminary experiments with a wider
range; however, only the ones showing significant effect at minimum
concentrations are reported. The zeta potential of LMP, measured at pH
3.0 using a zeta potential analyzer (Litesizer500, Anton Paar, Montreal,
QC, Canada), was -18.68 ± 0.2 mV. Control solutions with just S at
0.125, 0.5 and 1 wt\% and Ca at 0.045 wt\% were also prepared using
similar methods. All the solutions were prepared the same day as
emulsions. The oil phase was prepared by dissolving 4 wt\% GMO in canola
oil (CO) or mineral oil (MO) under stirring (400 rpm) at 60°C for 25
min.

2.3 Preparation of W/O emulsions

The aqueous and oil solutions were warmed at 40 ± 2 °C before weighing.
The oil phase (70 g, 80 wt\%) was weighed in a glass beaker (100 mL),
and the aqueous phase (17.5 g, 20 wt\%) was added dropwise while
stirring at 400 rpm. The same weight of each phase and similar size
glass beakers were used for all emulsion batches. Coarse emulsification
was done with a rotor-stator blender (Polytron, Brinkman, ON, Canada) at
level 10 for 1 minute. The coarse emulsions were then further
homogenized in a high-pressure homogenizer (Emulsiflex C3, Avestin Inc.,
Ottawa, ON, Canada) at 20,000 psi for three cycles. Preliminary
experiments showed that three cycles were enough to reduce the droplet
size, and after that, no further decrease was observed. The final
emulsions were collected in 250 mL beakers and cooled down at room
temperature while stirring with a magnetic stirrer (size 3.5 x 0.7 cm)
at 290 rpm for 20 minutes.

2.4 Emulsion storage stability

Freshly-made emulsions (25 mL) were transferred to 40 mL clear glass
vials (VWR, Edmonton, AB, Canada) and stored at room temperature for
seven days. The images were taken with an iPhone 8 camera (12 MP). The
images of the vials were used to measure the height of the emulsion
layer on day 0 and day 7 with ImageJ (v1.5 2i -- Fiji project) to
calculate the fraction of emulsion height (Eq.1).

\(Fraction\ of\ emulsion\ height=\frac{\text{Emulsion\ height\ }on\ day\ 7}{Total\ emulsion\ height\ on\ day\ 0}\)Eq. 1

2.5 Microstructure analysis

2.5.1 Light microscopy

The emulsion microstructure was captured after seven days of storage. A
drop of the emulsion was placed over a glass depression microscope slide
with 1 -- 1.2 mm thickness (VWR, Edmonton, AB, Canada) and covered with
a coverslip (VWR, Edmonton, AB, Canada). A light microscope (Eclipse
E400, Nikon Mississauga, ON, Canada) was used to observe the emulsion
samples using a 10x and 100x objective lens.

2.5.2 Confocal microscopy

Emulsion microstructure was also captured with a confocal microscope
(Nikon C2, Nikon Inc., Mississauga, ON, Canada). One drop of 0.1 wt\%
Nile red solution in mineral oil was mixed with nine drops of fresh
emulsion, and a drop of this mixture was placed over a glass depression
microscope slide covered with a coverslip and placed under the
microscope. The sample was excited using a 543 nm laser, and the
emission spectra were collected from 573 to 613 nm using a 40x and 60x
objectives lens.

2.6 Determination of emulsion droplet size

The droplet size of the emulsions was determined by analyzing the images
taken with the confocal microscope. It was essential to select multiple
images of each sample so that the number of water droplets quantified
was between 3,000 -- 18,000. The image analysis methodology developed in
Romero-Peña et al. (2020) was used to quantify the water droplet size
using ImageJ software (v1.5 2i -- Fiji project). The image analysis data
(area of each droplet) was exported to an Excel spreadsheet (Microsoft
Office 365), and further calculation was done to obtain the
volume-weighted means droplet diameter (\emph{d} \textsubscript{4,3})
using Eq. 2, where \emph{d} is the diameter of each droplet obtained
from the image analysis and \emph{n} is the number of droplets of a
particular size.

\(d_{4,3}=\frac{\sum_{1}^{n}d^{4}}{\sum_{1}^{n}d^{3}}\) Eq. 2

2.7 Accelerated stability analysis

The accelerated storage stability of fresh emulsions was analyzed using
an analytical photocentrifuge LUMiSizer\textsuperscript{®} LS650 (LUM
GmbH, Berlin, Germany) using the methodology used by Erramreddy et al.
(2017). An aliquot of emulsion (400 \selectlanguage{greek}μ\selectlanguage{english}L) was added with a syringe into a
rectangular polycarbonate cuvette (8 mm x 2 mm) (LUM GmbH, Berlin,
Germany) and centrifuged at 4000 rpm (2325xg). The equipment transmits
an 865 nm wavelength laser through the cuvettes every 60 s (1000
transmission profiles collected in 16 h 40 min) during the
centrifugation. The light intensity transmitted through the emulsion
depends on droplet movement under the centrifugal force. Data analysis
and calculation of sedimentation velocity (\selectlanguage{greek}μ\selectlanguage{english}m/s) from the final
transmission profile were processed using the SEPView\selectlanguage{ngerman}® software 4.1 (LUM
GmbH, Berlin, Germany).

2.8 Rheological properties of W/O emulsions

The apparent viscosity and viscoelasticity of fresh and 7 days old
emulsions were measured using an AR-G2 rheometer (TA Instruments,
Montreal, QC, Canada) at 25 °C with an acrylic parallel plate (60 mm
diameter). For viscosity measurement, a shear rate from 0.01 to 100
s\textsuperscript{-1} and a loading gap of 1000 \selectlanguage{greek}µ\selectlanguage{english}m were used. The
oscillatory mode was used at a strain ranging from 0.01 to 1000\% to
measure storage (G') and loss moduli (G'') at a constant frequency of 1
Hz. (6.18 rad/s). The apparent viscosity values of emulsions at a shear
rate of 0.1 s\textsuperscript{-1} on day 0 and 7 were used to calculate
the viscosity index (Eq. 3) to determine the change in viscosity with
time.

\(Viscosity\ index=\ \frac{\text{Apparent\ viscosity\ of\ emulsion\ }on\ day\ 7}{\text{Apparent\ viscosity\ of\ fresh\ emulsion}}\)Eq. 3

2.9 Determination of interfacial tension

A force tensiometer (K20 by Kr\selectlanguage{ngerman}üss GmbH, Hamburg, Germany) with a
Wilhelmy plate (20 x 10 mm) was used to determine oil/water interfacial
tension at room temperature. The oil phase (65 mL) with 4 wt\% GMO was
added in a wide-mouth glass beaker (130 mL). The Wilhelmy plate was
immersed entirely in the oil phase, set as the zero-immersion depth. The
plate was then removed and cleaned with acetone (A18-4, Fisher, US) and
flamed with a butane torch (Bernzomatic, OH, USA). The aqueous phase (22
mL) was added to another similar glass beaker and placed over the stage.
The plate was placed very close to the liquid-air interface for surface
detection. Then, the oil phase was gently added to the aqueous solution
with a pipette (VWR, Edmonton, AB, Canada) to fill the zero-immersion
depth. The interfacial tension measurements were performed until a
standard deviation of 0.1 mN/m could be reached between the readings.

2.10 Statistics

All emulsions were prepared in triplicate, and each of them was analyzed
on day 0 and day 7, and the average and standard deviation were
reported. The statistical analysis was performed using IBM SPSS
Statistics 20, in which Tukey's test was performed to compare the
average with a significance level of 0.05.

\par\null

\textbf{3. Results and discussion}

3.1 Emulsion storage stability after seven days

Phase separation in the W/O emulsions was visually observed and recorded
(Figure 1). After 7 days, a clear oil phase was separated at the top in
most emulsions due to water droplet sedimentation. Higher emulsion
height or lower oil phase separation indicates better emulsion
stability. In Figure 1, the control emulsions were indicated with
no-additive (S0). The emulsions with only salts are indicated as S0.125
(0.125\% sodium chloride) and Ca0.045 (0.045\% calcium chloride). For
the emulsions without any additive (S0), CO emulsions showed larger
phase separation (lowest stability) compared to MO emulsions (Figure 1A
and 1B). The addition of S0.125 showed a significant increase in
emulsions height compared to S0 in both bulk oils. The emulsions with
S0.125 and AA or CA demonstrated higher emulsion height and lower oil
separation for both CO and MO than those with only AA or CA. The
addition of Ca also increased emulsion height compared to S0 in both
oils (Figure 1C). The emulsions with only LMP reported a higher emulsion
height than those with LMP and Ca, indicating Ca was not that critical
for storage stability when LMP was present. Besides, LMP emulsions
(Figure 1C) seem to be more stable with lower phase separation for both
CO and MO than the emulsions formed with S-AA and S-CA (Figure 1A, B).
For most emulsions, lower phase separation was observed with MO than CO,
indicating higher emulsion stability with the MO, which followed the
observation by Ghosh et al. (2011).

3.2 Quantification of emulsion kinetic stability against phase
separation

The emulsion layer height was measured from Figure 1 and presented as a
fraction of the total emulsion height (Eq. 1) (Figure 2). A higher value
of emulsion height fraction signifies emulsions with higher stability
against phase separation. CO-emulsions height showed a significant
increase from S0 (0.16 ± 0.01) to S0.125 (0.54 ± 0.02) (Figure 2A).
Similarly, in MO emulsions, the height significantly increased from S0
to S0.125 (0.39 ± 0.01 to 0.72 ± 0.01). For both CO and MO emulsions,
only AA and CA (both 0.125\% and 0.5\%) did not improve the emulsion
height further than the S0.125, which indicates that the organic acids
added to the aqueous phase were not as effective as salt in improving
emulsion stability. Interestingly, when both salt and organic acids were
present, the CO-emulsions height significantly increased compared to
only salt-added emulsions. In contrast, for MO-emulsion, no significant
difference was observed when both salt and organic acids were present
compared to only salt (Figure 2A). For both the CO and MO emulsions,
S0.125 with either AA or CA showed no significant difference in emulsion
height fraction considering the same oil phase (Figure 2A).

With the addition of LMP (both 1.5 and 2\%) (Figure 2B), a significant
improvement in both CO and MO emulsion stability was observed. It is
possible that the acidic aqueous phase (pH 3) induced structural opening
in the pectin (Schmidt et al., 2015), which could have resulted in
interfacial interaction with GMO polar head groups, thereby stabilizing
GMO at the interface and increasing emulsion stability. The emulsion
height reached the highest in MO-emulsions with LMP, although the values
in CO-emulsions with LMP were not significantly different. However, as
soon as Ca was added to the LMP-containing CO-emulsions, the height
decreased to 0.67 ± 0.02 and 0.61 ± 0.02 for LMP1.5 and LMP2,
respectively. It could be possible that Ca formed ionic linkages between
the carboxylic groups from two LMP chains, which decreased LMP
interaction with GMO at the interface leading to an increase in emulsion
destabilization (Flutto, 2003). In MO-emulsions, 1.5 \% LMP with Ca
demonstrated reduced emulsions height (0.66 ± 0.02). Conversely, 2 \%
LMP with Ca did not significantly diminish emulsions height (0.75 ±
0.02) than only LMP-added MO-emulsions. It might suggest that for
LMP2-Ca, excess unreacted LMP was available to stabilize the interfacial
GMO leading to better emulsion stability. Between CO and MO emulsions,
it can be said that the effectiveness of LMP in improving liquid W/O
emulsion stability was hindered by Ca addition, more extensively in CO
than MO.

3.3 Emulsion microstructure

All emulsions formed water droplets dispersed in the continuous oil
phase as a free and aggregated network. The control CO emulsions without
any additive (S0) showed a few larger droplets with some aggregation as
most droplets were destabilized phase-separated (Figure 3A). The
incorporation of S0.125 showed the initial appearance of extensive water
droplet network formation (dark irregular-shape matter in the image)
(Figure 3B). Such droplet aggregation also appeared with AA or CA
addition into the aqueous phase (Figure 3C, D and G, H). When S0.125\%
was incorporated with AA (Figure 3E, F) and CA (Figure 3I, J), a more
extensive water droplet network structure was identified, where free
droplets were hard to locate in the continuous phase. It can be said
that the water droplet network's extension was responsible for their
higher emulsion height (Figure 1 and 2). Similar water droplet
aggregation was also observed for W/O emulsions stabilized with Span 80
and lecithin emulsifiers (Ushikubo \& Cunha, 2014).

MO-emulsions without any aqueous phase additive (S0) showed water
droplets well distributed in the continuous phase (Figure 4A). Similar
to CO emulsion, the microstructure of MO-emulsions with S0.125 (Figure
4B), AA (Figure 4C, D) or CA (Figure 4G, H) also demonstrated a water
droplet network formation. The addition of S0.125\% with AA or CA in
MO-emulsions developed an extensive water droplets network structure
similar to CO-emulsions.

The microstructure of emulsions formed with LMP and Ca are presented in
Figure 5. CO-emulsions without Ca (S0, Figure 5 I-A) and with Ca (Figure
5 I-B) showed dispersed water droplets with some degree of droplet
coalescence. The incorporation of LMP formed an extensive water droplets
network throughout the emulsion, making it challenging to capture the
emulsion's clear images (Figure 5 I-C, I-D). However, the mixture of LMP
and Ca illustrated a different structure related to gel-formation due to
the interaction between calcium ions and the carboxyl groups in the
pectin molecules (Yuliarti \& Othman, 2018). The structure formed by
LMP-Ca in the aqueous phase can coexist with the water droplet network
(Figure 5 I-E, I-F). All MO-emulsions, in the presence of LMP (with or
without Ca) (Figure 5 II-B, II-A), also formed an extensive water
droplets network compared to the emulsions without LMP (Figure 5 II-C,
II-D). Comparing the CO and MO-emulsions with LMP, it can be said that
the presence of MO led to a finer droplet network that was better able
to provide emulsion stability against phase separation. Overall, CO and
MO-emulsions with LMP (Figure 5) showed a more expanded water droplets
network of smaller water droplets than the emulsions with S, AA, CA, or
their mixture (Figures 3 and 4). Such droplet networks could be
responsible for increasing emulsion stability against phase separation
reported in Figures 1 and 2.

3.4 Average water droplet size

The water droplet size was calculated using image analysis. Only the
water droplets outside the network structure were analyzed for this
calculation. Thus, emulsions with LMP and Ca in CO and MO were not
reported because the water droplets were aggregated, forming a network
spanning the whole microstructure. It was assumed that the free water
droplet size would represent the entire emulsions for comparison
purposes. The control CO emulsions (S0) showed a high
d\textsubscript{4,3} (9.2 ± 0.1 \selectlanguage{greek}µ\selectlanguage{english}m), which was similar to CA0.5,
followed by CA0.125 (8.2 \selectlanguage{ngerman}± 0.0 \selectlanguage{greek}µ\selectlanguage{english}m) and S0.125 (7.9 \selectlanguage{ngerman}± 0.0 \selectlanguage{greek}µ\selectlanguage{english}m) (Figure
6A). The emulsions formed with AA showed considerably lower
d\textsubscript{4,3} (6.0 \selectlanguage{ngerman}± 0.1 \selectlanguage{greek}µ\selectlanguage{english}m) at both concentrations. The addition
of S to either AA or CA showed the smallest droplet size of all CO
emulsions, which could be related to their higher emulsion height
fraction reported in Figure 2. All MO emulsions reported a much smaller
droplet size (about 7 times lower) than CO emulsions (Figure 6B). The MO
emulsions prepared with S0 and S0.125 showed a non-significant
difference in d\textsubscript{4,3} (1.2 \selectlanguage{ngerman}± 0.03 \selectlanguage{greek}µ\selectlanguage{english}m). However, the
incorporation of AA and CA and their mixture with S reported a
significant reduction of droplet size (\textless{} 0.72 \selectlanguage{ngerman}± 0.01 \selectlanguage{greek}µ\selectlanguage{english}m). The
smaller droplet size obtained in MO emulsions compared to CO-emulsions
can be a factor for their higher emulsion stability against phase
separation (Figure 1 and 2).

3.5 Accelerated sedimentation velocity of emulsions

Water droplets' sedimentation velocity under accelerated gravitation
(2325xg) was used to identify how fast the emulsions would phase
separate (Figure 7), which was used as an indicator of long-term
stability. The CO emulsions with S0 and S0.125 had high sedimentation
velocity under accelerated gravitation (on average, 9.8 \selectlanguage{ngerman}± 0.2 \selectlanguage{greek}µ\selectlanguage{english}m/s),
which could be due to the larger water droplets and coalescence under
centrifugal force (Figure 7A). There was a sharp decrease in
sedimentation velocity with the addition of AA, CA, or their mixture
with S, which can be associated with the combined presence of smaller
water droplets and the droplet network formation.

The sedimentation velocity in MO emulsions significantly decreased with
S0 (6.8 \selectlanguage{ngerman}± 0.2 \selectlanguage{greek}µ\selectlanguage{english}m/s) and S0.125 (7.3 \selectlanguage{ngerman}± 0.1 \selectlanguage{greek}µ\selectlanguage{english}m/s) compared to
CO-emulsions, which could be due to the smaller droplet size of the
former (Figure 7A). However, the sedimentation velocity of all other MO
emulsions with AA, CA, S-AA, or S-CA demonstrated higher values than CO
emulsions, despite their visually higher emulsion height, under earth
gravitation after seven days (Figure 1 and 2). It can be said that the
water droplet network provided better emulsion kinetic stability against
earth gravity. However, accelerated gravitational force probably broke
the network structure, leading to a faster separation of large water
droplet aggregates in MO-emulsions. Erramreddy et al. (2017) reported a
correlation between gel yield point and centrifugal separation. The
minimum relative centrifugal force required for the creaming of oil
droplets increased with gel strength. In the present case, the higher
sedimentation velocity of MO-emulsions under accelerated gravitation
could be due to their lower-yielding behaviour than CO emulsions, which
will be discussed under the viscoelasticity section.

The addition of Ca significantly reduced CO-emulsion sedimentation
velocity (7.4 \selectlanguage{ngerman}± 0.4 \selectlanguage{greek}µ\selectlanguage{english}m/s) compared to S0, indicating higher stability
against phase separation (Figure 7B). Emulsions with LMP (both
concentrations) exhibited a further reduced sedimentation velocity.
Similar behaviour was also observed for MO emulsions, except for the one
with Ca. Overall, CO-emulsions with AA, CA, their mixtures with S and
LMP exhibited lower sedimentation velocity than MO-emulsions. Lower
sedimentation velocity indicates that the presence of aqueous phase
ingredients could help to improve long-term W/O emulsion stability.

3.5 Change in emulsion viscosity with time

The emulsion viscosity changes were calculated using the viscosity
index, a ratio of apparent emulsion viscosities on day 7 to day 0
(Figure 8). A value of viscosity index less than one indicates that the
viscosity decreased upon storage, which could be due to the water
droplet networks breakdown and lowering of emulsion stability against
phase separation. All CO-emulsions reported viscosity indices less than
one, except the emulsions with S-CA, where they remained close to one
(Figure 8A). Conversely, all MO-emulsions with additives in the aqueous
phase reported a viscosity index above one, except the S-CA emulsions,
which can strengthen the water droplet network structure with time,
leading to higher stability of MO-emulsions.

With LMP, all CO-emulsions showed viscosity indexes lower than one,
except by LMP2-Ca0.045, which had the highest viscosity index (1.48 \selectlanguage{ngerman}±
0.02), which could be associated with the strongest network formation
after storage (Figure 7B). On the contrary, all MO emulsions with LMP
demonstrated a viscosity index very close to one (Figure 7B). Overall,
the MO bulk phase might allow more interaction of the aqueous phase
additives with GMO at the interface due to the less interaction of GMO
with MO hydrocarbon. This way, higher droplet stability and a more
robust droplet network could be developed during storage, leading to
better viscosity protection for MO emulsions than CO-emulsions.

3.6 Emulsion viscoelasticity

All the emulsions reported higher storage (G') than loss (G'') moduli in
the low-strain regime (Figures 9 and 10). No emulsions showed true
linear viscoelastic region (LVR) except fresh S0 (Figures 9 A1 day 0),
indicating weak gels. All emulsions showed a steady decline in G' with
an increase in strain, also indicating a weak gel structure.

After storage, all CO-emulsions reported lower range G' than the fresh
emulsions (Figure 9, A2), implying that the initial structure on day 0
has diminished in strength, similar to the lower viscosity index
reported in Figure 8. Most CO-emulsions showed on day 0 showed a peak in
G'' at the crossover, which indicates relaxation in droplet network
structure, leading to gel breakdown. However, such peak in G'' mostly
disappeared after 7-das storage, except for S-CA (both concentrations),
indicating loss of network structure with time. All MO-emulsions (Figure
9, B) exhibited a faster drop in G' with strain and lower crossover
stain than CO-emulsions, which could explain their higher sedimentation
velocity under accelerated gravitational force reported in Figure 7.
After 7 days, most MO-emulsions, except S-CA, showed unchanged or some
increase in G' (Figure 9, B1), which was also similar to the viscosity
index data (Figure 8)

Unlike CO-emulsions with S and AA or CA, no peak in G'' was observed
with Ca and LMP. After 7 days, all CO-emulsions showed mostly unchanged
weak viscoelastic behaviour (Figure 10, A2). Similar to what was
observed in Figure 9, in Figure 10, MO emulsion showed a faster drop in
G' as a function of strain and a crossover at a lower strain compared to
CO-emulsions. After storage, MO-emulsions with Ca and LMP1.5
demonstrated an increase in gel strength (Figure 10, B2). Overall, CO
and MO emulsions' viscoelastic behaviour showed different levels of weak
gel strength, with MO emulsions appearing weaker than the CO emulsions.
The presence of S, AA, CA or their combination in CO-emulsion created
gels that can withstand more shear force than the CO-emulsions with Ca
and LMP.

\par\null

3.7 Interfacial tension between oil and aqueous phases and average
droplet size

Toil-aqueous phase interfacial tension was determined to understand the
GMO's surface activity in the presence of various aqueous phase
additives. CO without GMO against water without any additive reported an
interfacial tension of 21.23 ± 0.05 mN/m, while for MO, GMO's
interfacial tension was 46.03 ± 0.73 mN/m (data not shown). According to
Tuntiwiwattanapun et al. (2013), vegetable oils with water displayed
interfacial tension between 20 -- 30 mN/m. With MO-water, Xaxa (2014)
reported an interfacial tension of 45.65 ± 0.57 mN/m. Both the values
from the present work are within the range found in the literature.
Although made up of non-polar TAG, commercial vegetable oils reduced the
interfacial tension due to minor polar components, such as free fatty
acids, monoacylglycerols, or diacylglycerols (Gaonkar, 1989).

Interfacial tension measurement relied on the emulsifier diffusion and
adsorption to the W-O interface. A higher concentration of
surface-active molecules should lower interfacial tension unless the
critical micelle concentration (CMC) has reached, after which no further
decrease in interfacial tension would be observed. Figure 11 presents
the interfacial tension of GMO in CO and MO against the various aqueous
phases. All GMO interfacial tension for the CO-aqueous phase showed a
non-significant difference (1.2 to 1.7 mN/m), and they were lower than
all MO-aqueous phase interfacial tension (1.4 to 2.5 mN/m), except for
S0, S0.125 (Figure 11A), and Ca0.045 (Figure 11B). Ghosh and Rousseau
(2012) reported the interfacial tension of 4 wt\% GMO in CO-water (1.6 ±
0.1 mN/m), which is similar to our data (1.2 ± 0.05 mN/m). GMO in MO-S0
system displayed a lower interfacial tension, which can be associated
with GMO's ability to pack much better at the interface against MO.
Lower interfacial tension of GMO in MO-aqueous phase without any
additives was also responsible for the much smaller water droplet size
in W/MO emulsion (0.7 to 1.2 \selectlanguage{greek}μ\selectlanguage{english}m) than W/CO emulsions (4.6 to 9.2 \selectlanguage{greek}μ\selectlanguage{english}m)
(Figure 6).

The addition of S, AA, CA or their mixtures did not significantly change
W-CO interfacial tension. In the W-MO system, with the addition of
sodium chloride (S0.125), a slight increase in interfacial tension was
observed. Amar-Yuli and Garti (2005) reported that S creates a
''salting-out'' effect, reducing the emulsifier head group
hydrophilicity (due to dehydration) and promoting aggregation. The
salting-out outcome may be favourable in oils with excess surface-active
molecules, as in vegetable oils. However, in MO, the increase of GMO
interfacial aggregation might promote some bare patches at the
interface, which may not favour a decrease in interfacial tension.
Besides, Amar-Yuli et al. (2007) addressed that the hydration layer of
GMO polar heads decreased in size due to dehydration when GMO competes
with other molecules for binding water, leading to an increase in
interfacial tension. MO-aqueous phase system also observed a significant
increase in interfacial tension with AA and CA compared to the aqueous
phase without any additive. Therefore, increased emulsion stability
against phase separation with AA and CA was not due to interfacial
tension (their droplet size was also similar), but rather water droplet
aggregation and network formation.

GMO in CO-water with Ca and LMP showed similar interfacial tension to
the aqueous phase without additive (S0) (Figure 11B). Lutz et al. (2009)
investigated the interfacial tension of limonene-water with 0.5 wt\%
pectin. They attributed the enhancement of GMO orientation at the
interface due to the interaction of pectin carboxylic groups.
Interfacial tension of the MO-aqueous phase with LMP showed an increase
compared to the MO-aqueous phase without any additive or with Ca (Figure
12B). It could be due to the presence of LMP at the O/W interface (due
to the lack of surface active molecule in MO) preventing GMO's free
adsorption at the interface. Many researchers have previously reported
the surface activity of LMP. For example, Leroux et al. (2003) reported
a decrease in interfacial tension between paraffin oil and 2 wt\% pectin
solution with a reduction in the degree of methylation. The addition of
Ca\textsuperscript{2+} to LMP in the aqueous phase led to a steep rise
in GMO's interfacial tension for both the CO and MO emulsions. Calcium
ions can promote gelation in LMP molecules due to their ionic binding
with pectin carboxylic groups (Capel et al., 2006). LMP formed weak gels
in the aqueous phase at room temperature with Ca\textsuperscript{2+},
which might reduce GMO adsorption at the interface, leading to increased
interfacial tension. Higher interfacial tension of LMP-Ca systems can
also be related to their reduced emulsion stability (61 -- 67\%)
compared to only LMP-added systems (72\%) (Figure 2). Overall, the
interfacial tension was influenced by the oil type and the interfacial
interaction between the GMO head group and the aqueous phase additives.
Interestingly, interfacial tension had less influence on emulsion
stability against phase separation, which was mostly influenced by
droplet aggregation and network formation.

\textbf{3.8 Discussion}

We have shown that incorporating various aqueous phase ingredients
interacted with GMO differently at the W-O interface, leading to
improved W/O emulsion stability against phase separation for both TAG
(in CO) and paraffin hydrocarbon (in MO)-based oils. The lowest
stability was observed for the emulsions without any aqueous phase
additives. Incorporation of either sodium chloride (S) or calcium
chloride (Ca) showed significant improvement in emulsion sedimentation
stability. Salt-induced improvement in W/O emulsion stability has been
well studied, and many authors reported that the presence of salt in the
aqueous phase is essential for efficient stabilization of W/O emulsions
(Ganguly et al., 1992; M\selectlanguage{ngerman}árquez et al., 2010; Opawale \& Burgess, 1998;
Scherze et al., 2006). It has been proposed that salt improves non-ionic
emulsifier interfacial adsorption density, improving interfacial
elasticity and emulsion stability (Aronson \& Petko, 1993). Besides
salts, we have also added organic acids such as ascorbic acid (AA) and
citric acid (CA) into the aqueous phase, which also showed significant
improvement in W/O emulsion stability compared to the emulsions without
any additives. It has been proposed that an ideal emulsifier for W/O
emulsion must form a strong hydrogen bond with water with its polar head
group (Villamagna et al., 1995). However, in canola oil (CO), GMO form
stronger hydrogen bond with \textgreater{}C=O groups of TAG glycerol
polar heads compared to the -OH groups of water, leading to an eventual
disruption of GMO molecules from the W-O interface towards the oil phase
(Ghosh et al., 2011). The addition of AA or CA with multiple
\textgreater{}C=O groups in the aqueous phase provided stronger hydrogen
bonds with interfacial GMO towards the aqueous phase, thereby providing
improved stability to the W/O emulsions. Such interaction of GMO with
ascorbic acid in water has previously been reported by
Bitan-Cherbakovsky et al. (2009). Improvement in W/O emulsion stability
in the presence of AA and CA was also observed for mineral oil (MO).
Interestingly, a synergistic improvement in emulsion stability was
observed when both S and AA or S and CA were added to the aqueous phase.
Such synergistic effect in increasing W/O emulsion stability was not
reported before. Such differences in emulsion stability can also be seen
from their average droplet size. For CO-emulsions, the smallest droplet
size was observed when both S and AA or S and CA were present in the
aqueous phase. Comparing CO and MO, a much smaller average droplet size
and higher sedimentation stability were observed for MO-emulsions
compared to CO-emulsions. This could be due to a lack of interactions
between GMO and MO, leading to a favourable presence of GMO at the W-O
interface than when GMO was present in CO. Such difference in W/O
emulsion stability based on the continuous oil phase has also been
reported before (Bus et al., 1990; Ghosh \& Rousseau, 2011). For
example, Bus et al. (1990) showed that if the emulsifier does not form
any hydrogen bond with the oil (as in the case of GMO in MO), they will
be more available to form hydrogen bonds with water, and their stability
will be higher compared to a hydrogen-bonding oil such as CO.

We have also investigated the presence of polysaccharide pectin (LMP)
with numerous \textgreater{}C=O groups and -OH groups on the stability
of W/O emulsions. Our initial hypothesis was that the presence of a
large number of hydrogen bond-forming groups in the aqueous phase would
provide extensive stability to GMO at the W-O interface. We found the
highest emulsion stability in the presence of LMP in the aqueous phase.
Moreover, the hydrogen bonding ability of LMP with GMO was able to
overcome the destabilization effect of CO on interfacial GMO, so that
when LMP was present, no significant difference in the stability of the
W/CO and W/MO emulsions was observed. However, the presence of Ca and
LMP led to lower emulsion stability, which was due to the interaction of
Ca with LMP making it less available to hydrogen bond with interfacial
GMO. The effect of pectin on improving W/O emulsion stability has
recently been reported for W/CO emulsion-based low-fat tablespread by
Romero-Peña and Ghosh (2021).

The microstructure of the emulsions revealed extensive aggregations of
water droplets and the formation of a droplet network in both oils. It
is known that water droplets in W/O emulsion generally have a smaller
energy barrier against flocculation (Kent \& Saunders, 2001), which
could also facilitate droplet aggregation. It can be said that the
extensive water droplet network was eventually responsible for the
higher stability of the W/O emulsions against sedimentation. For
example, water droplets containing LMP aggregated massively, more than
any other additives, which could be related to the interaction between
GMO and LMP and interfacial strengthening of water droplets surfaces
leading to the highest emulsion stability (Romero-Peña \& Ghosh, 2021).

From the viscosity index, it was observed that while the water droplet
network structure was protected and indices remained \selectlanguage{english}[?] 1 for all
MO-emulsions, for most of the CO-emulsions viscosity, dropped with
storage time, indicating breakdown of droplet network with time.
Viscoelastic behaviour of all emulsions showed weak gel-like properties.
Interestingly, all MO-emulsions showed a faster drop in storage moduli
with strain and lower crossover strain than the CO-emulsions. A similar
indication of weaker droplet network structure for MO-emulsions was also
observed when the emulsions were subjected to accelerated gravitation,
where all MO-emulsion showed faster sedimentation velocities due to the
faster breakdown of water droplet network in MO than that in CO.
However, with LMP in the aqueous phase, such a difference in
sedimentation velocity was smaller, which could be due to the
enhancement of the droplet network in the presence of LMP. More research
is needed to further understand the mechanism behind the difference in
water droplet network strength in these two different oils

The excess of polar molecules in CO reduced the interfacial tension to a
minimum which did not change in the presence of any aqueous phase
ingredients. However, this mechanism was not enough to stabilize the CO
emulsions without additives (S0) due to the stronger hydrogen bonds
between GMO and TAG glycerol polar head groups of CO, which pulled the
GMO out of the interface towards the oil phase in an emulsified state
(Bus et al., 1990; Ghosh et al., 2011). It can be said that the
interfacial tension determined using a platinum plate in a static setup
was not able to simulate the dynamics in play at the water droplet
interface in the presence of various aqueous phase additives and the oil
phase. When GMO was present in MO, interfacial tension was the lowest
without any aqueous phase additive, further supporting the hypothesis of
lack of hydrogen bonding of GMO in MO leading to their free movement
towards the interface. Interestingly, although the interfacial tension
of GMO in MO was higher than GMO in CO in the presence of various
aqueous phase additives, the droplet size of the final emulsions was
significantly smaller for MO-emulsions than CO-emulsions. This apparent
discrepancy could be explained by partial droplet destabilization in CO
due to GMO's disruption towards the oil phase. Finally, LMP molecules
demonstrated better stability of GMO and similar interfacial tension in
both oils due to its numerous \textgreater{}C=O groups that could form
hydrogen bonds with interfacial GMO. Although the interfacial tension in
the presence of LMP was not significantly different compared to AA, CA
or their mixture with S, LMP provided a stronger interface which allowed
higher stability of the droplets in an aggregated state (Ford \&
Furmidge, 1966), thereby improving emulsion stability and rheology.
However, adding Ca ions with LMP led to the formation of weak gel in the
aqueous phase, which hindered GMO adsorption at the O-W interface and
reduced emulsion stability.

\textbf{4. Conclusions}

In this work, different concentrations of hydrogen bond-forming agents
(citric acid (CA), ascorbic acid (AA), low methoxyl pectin (LMP)) with
and without salts (sodium chloride (S) or calcium chloride (Ca)) was
added into the aqueous phase to investigate the stability of the liquid
W/CO and W/MO emulsions with a small molecule emulsifier, GMO. W/CO
emulsion without additives destabilized instantly, whereas W/MO emulsion
stayed partially stable due to extensive hydrogen bonds between GMO and
CO TAG glycerol polar heads, leading to the desorption of interfacial
GMO towards the CO phase. In MO, GMO favoured interfacial adsorption due
to the lack of polar groups, leading to emulsion stability. The addition
of hydrogen bond-forming agents and salts in the aqueous phase
significantly improved both the CO- and MO-emulsion stability. LMP, with
a large number of hydrogen bond-forming groups, was able to provide the
highest emulsion stability after 7 days in both oils compared to AA, CA
and their mixtures with S. However, the presence of Ca with LMP led to
gelation in the aqueous phase which prevented interfacial interaction of
LMP with GMO leading to a lowering of emulsion stability. Emulsions with
both oils formed weak gels due to the formation of an extensive network
of water droplet aggregates. Water droplets were smaller in MO-emulsions
than CO-emulsions, leading to a more extensive droplet network in the
former which provided higher stability against phase separation and
preservation of viscosity after 7 days storage. Interestingly, the
droplet network in MO-emulsions broke down at a lower crossover stain
than the CO-emulsions, indicating weaker links between the droplets.
Overall, the aqueous phase hydrogen bond-forming agents interacted with
GMO at the interface allowing improved stability of GMO-stabilized
liquid W/CO emulsions. This important finding could facilitate the
development of highly stable food-grade low-saturated fat tablespreads.
The knowledge developed could also improve our understanding of
stability mechanisms in W/O emulsions for other industrial and cosmetics
applications.

\textbf{Acknowledgements}

This research was funded by the NSERC Discovery Grant, Canada Foundation
for Innovation (CFI) Leaders Opportunity Fund and the Saskatchewan
Innovation and Science Fund (ISF).

\textbf{Conflict of interest statement}

The authors declare no conflict of interest.

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