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\begin{document}

\title{The effect of addition of high-melting monoacylglycerol and candelilla
wax on pea and faba bean protein foam-templated oleogelation}



\author[1]{Athira Mohan}%
\author[2]{Michael Nickerson}%
\author[3]{Supratim Ghosh}%
\affil[1]{University of Saskatchewan College of Agriculture and Bioresources}%
\affil[2]{USASK, Canada}%
\affil[3]{University of Saskatchewan}%


\vspace{-1em}



  
  \date{May 26, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Oleogels prepared from hydrocolloids have recently gained a lot of
attention as an alternative for trans and saturated fats. Previously we
have demonstrated that the freeze-dried foams prepared using a mixture
of 5\% faba bean or pea protein concentrates with 0.25\% xanthan gum at
pH 7 and 9 can hold canola oil 30-40 times their weights (Mohanan, Tang,
Nickerson and Ghosh, 2020). However, the oleogels suffered from high oil
loss, about 30\% oil leaked, which negatively affected the rheological
properties of the oleogels. The functionality of the cake baked using
the oleogels was poorer compared to a shortening baked cake. The present
study explored the addition of a small amount of high-melting
monoacylglycerol (MAG) and candelilla wax (CW) on reducing oil loss,
improving rheological properties and baking qualities of pulse
protein-stabilized oleogels. Different concentrations (0.5-3\%) of MAG
or CW were dissolved in canola oil at 80 \selectlanguage{ngerman}ºC. The hot oil was then added
into the freeze-dried protein-polysaccharide foams (pH 7) and quickly
transferred to a refrigerator. The crystallized additives reinforced the
oleogel network, thereby reducing oil loss while increasing the
firmness, cohesiveness, and storage modulus. When model cakes were baked
with the oleogels, significant improvement in textural properties was
observed with the addition of MAG in the foam-templated oleogels.
However, in comparison with shortening-based cakes, oleogel-based cakes
still showed a negative effect on hardness, chewiness and cohesiveness.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{The effect of addition of high-melting monoacylglycerol and
candelilla wax on pea and faba bean protein foam-templated oleogelation}

Athira Mohanan, Michael T. Nickerson, and Supratim Ghosh*

University of Saskatchewan, Saskatoon, SK, Canada



\section*{}

{\label{section-1}}

Short running title:

Pulse protein foam-templated oleogels

*Corresponding author

Supratim Ghosh, Associate Professor

Department of Food and Bioproduct Sciences, University of Saskatchewan

51 Campus Drive, Saskatoon, SK S7N5A8, Canada

Email: supratim.ghosh@usask.ca

Ph: 1-306-966-2555

\section*{Abstract}

{\label{abstract}}

Oleogels prepared from hydrocolloids have recently gained a lot of
attention as an alternative for trans and saturated fats. Previously we
have demonstrated that the freeze-dried foams prepared using a mixture
of 5\% faba bean or pea protein concentrates with 0.25\% xanthan gum at
pH 7 and 9 can hold canola oil 30-40 times their weights (Mohanan, Tang,
Nickerson and Ghosh, 2020). However, the oleogels suffered from high oil
loss, about 30\% oil leaked, which negatively affected the rheological
properties of the oleogels. The functionality of the cake baked using
the oleogels was poorer compared to a shortening baked cake. The present
study explored the addition of a small amount of high-melting
monoacylglycerol (MAG) and candelilla wax (CW) on reducing oil loss,
improving rheological properties and baking qualities of pulse
protein-stabilized oleogels. Different concentrations (0.5-3\%) of MAG
or CW were dissolved in canola oil at 80 \selectlanguage{ngerman}ºC. The hot oil was then added
into the freeze-dried protein-polysaccharide foams (pH 7) and quickly
transferred to a refrigerator. The crystallized additives reinforced the
oleogel network, thereby reducing oil loss, while increasing the
firmness, cohesiveness, and storage modulus. When model cakes were baked
with the oleogels, significant improvement in textural properties was
observed with the addition of MAG in the foam-templated oleogels.
However, in comparison with shortening-based cakes, oleogel-based cakes
still showed a negative effect on hardness, chewiness and cohesiveness.

\textbf{Keywords} : pulse proteins; foam-templated oleogel;
monoacylglycerol; candelilla wax; cake baking; texture profile analysis

\section*{Introduction}

{\label{introduction}}

Self-standing solid-like structures from liquid vegetable oil, the
so-called oleogels, has recently received significant attention due to
its potential to replace trans-fat and reduce saturated fats in
confectionery, baked products, and processed meats (Marangoni, Van
Duynhoven, Acevedo, Nicholson and Patel, 2020) In conventional oleogels,
the structure-forming agents are typically small molecule oleogelators,
such as plant waxes, fatty esters, monoacylglycerols, and ethyl
cellulose (Patel and Dewettinck, 2016, Singh, Auzanneau and Rogers,
2017). However, with more attention towards `clean label' and `healthy'
alternatives, the use of food hydrocolloids such as gums, and proteins
as oleogelators has recently gained more consideration (Doan, Van de
Walle, Dewettinck and Patel, 2015, Gravelle, Blach, Weiss, Barbut and
Marangoni, 2017, Gravelle and Marangoni, 2018, Lim, Hwang and Lee,
2017). However, these hydrophilic biopolymers are unable to form a
crosslinked network via intermolecular interactions if directly added
into the oil; therefore, indirect approaches are required to form a
structured oil phase. DeVries and coworkers successfully converted whey
protein stabilized hydrogels into oleogels by a step-wise solvent
exchange process (de Vries, Hendriks, van der Linden and Scholten, 2015,
de Vries, Wesseling, van der Linden and Scholten, 2017). \selectlanguage{greek}κ-\selectlanguage{english}Carrageenan
stabilized hydrogels were also converted into aerogels using
supercritical carbon dioxide and then into oleogels by allowing the
aerogel to adsorb liquid oil (Manzocco, Valoppi, Calligaris, Andreatta,
Spilimbergo and Nicoli, 2017). Combinations of surface active and
non-surface active biopolymers, such as hydroxypropyl methylcellulose
(HPMC) or methylcellulose (MC) with xanthan gum (Patel, Cludts, Bin
Sintang, Lewille, Lesaffer and Dewettinck, 2014); and soy protein
isolate with \selectlanguage{greek}κ-\selectlanguage{english}carrageenan (Tavernier, Patel, Van der Meeren and
Dewettinck, 2017) were also used to develop emulsion-templated oleogels
by removing the water using freeze-drying or vacuum-drying process.
Patel and coworkers were the first to use an HPMC foam-templated
approach to obtain oleogels (Patel, Schatteman, Lesaffer and Dewettinck,
2013). The freeze-dried HPMC foam was able to hold an oil 98 \% of the
foam weight. However, one of the most popular biopolymers, pulse
proteins, such as those from pea, lentil and faba bean have not been
explored significantly for oleogelation, despite their nutritional and
functional quality and higher consumer acceptance.

Our recent studies demonstrated that freeze-dried foams stabilized by
combinations of a pea or faba bean protein concentrates with xanthan gum
(XG) can be used to structure canola oil (CO) (Mohanan, Tang, Nickerson
and Ghosh, 2020). The foams prepared with 5 wt\% faba bean protein
concentrate (FPC) or pea protein concentrate (PPC) with 0.25 wt\% XG at
pH 7 and pH 9 were able to hold an oil more than 20 -- 30 times of their
weight. However, about 30-40\% of the added oil was leaked out of the
oleogel upon centrifugation (Mohanan, Tang, Nickerson and Ghosh, 2020).
Due to the poor oil binding capacity of the protein foam-templated
oleogels, their rheological properties were unreliable. Higher oil
leakage during rheology measurements of the oleogels led to a
significantly higher storage modulus (G') compared to the oleogels with
higher oil content (or higher oil binding capacity). Also, the baking
quality of the cakes baked using the oleogels was poor due to
significantly higher hardness and chewiness compared to a cake baked
using conventional high-melting shortening (Mohanan, Tang, Nickerson and
Ghosh, 2020). Therefore, the objective of the present study was to
improve the oil binding properties of the pulse protein foam-templated
oleogels using small quantities of conventional oleogelators, such as
high-melting monoacylglycerols (MAG) and candelilla wax (CW). We
hypothesized that the oleogelators would act as fillers in the protein
network to reduce the oil loss (OL) and improve the rheology of the
oleogels and therefore improve the textural properties of the cakes. The
overall goal was to see how much of the conventional high-melting
shortening functionality can be mimicked by the pulse protein-based
oleogels.

\section*{Experimental Procedures}

{\label{experimental-procedures}}

\subsection*{Materials}

{\label{materials}}

Pea protein concentrate (PPC, PP55) and Faba bean protein concentrate
(FPC, FBP60) were kindly donated by AGT Food and Ingredients (Saskatoon,
SK, Canada). Proximate analysis of the pulse protein samples is given in
Table S1 (Supporting Information). The protein content of PPC and FPC
was 51.4 and 57.6 wt\%, respectively. Gluten-free xanthan gum (XG) was
purchased from Bulk Barn store, a local supplier of Duinkerken Foods
Inc. (Slemon Park, PE, Canada). Vegetable shortening (Crisco brand,
composed of soybean oil, hydrogenated palm oil, modified palm oil, mono
and diglycerides, TBHQ and citric acid) and canola oil (Great Value
brand) were purchased from Walmart supercentre (Saskatoon, SK, Canada).
Refined candelilla wax was donated by Multiceras (Monterrey, NL,
Mexico). Powdered monoacylglycerol (MAG, product code DMG0093) was
donated by Palsgaard (Palsgaard Industry de Mexico, San Luis, S.L.P.,
Mexico). According to the supplier analysis report, the mixture of
monoacylglycerols contained about 37\% glycerol mono-stearate, 54\%
glycerol mono-palmitate, and 7.5\% free fatty acids with a melting point
\textasciitilde{}70 \selectlanguage{ngerman}ºC. Deionized water (Synergy~UV Water Purification
System, Millipore Sigma, Oakville, ON, Canada) was used for all the
solution preparation. Sodium azide and all other chemicals were
purchased from Sigma Aldrich Canada (Oakville, ON, Canada).

\subsubsection*{Foam preparation}

{\label{foam-preparation}}

Foams were prepared using a mixture of 5 wt\% protein (PPC or FPC) and
0.25 wt\% XG at pH 7, according to Mohanan, Nickerson and Ghosh (2020).
Protein and XG solutions were prepared separately and stirred overnight
at room temperature for proper mixing. Sodium azide (0.02 wt\%) was
added to the solutions to prevent microbial growth. The required amount
of protein and XG solutions were mixed to make up a volume of 400 mL and
stirred using a magnetic stirrer (400 rpm) for 30 min. The pH was then
adjusted to 7 using 1 M NaOH prior to foam preparation. A KitchenAid
Ultra Power Mixer (KitchenAid, Whirlpool Canada, Mississauga ON) with a
4.5 qt (4.3 L) stationary bowl and stainless-steel rotating beaters were
used for foam formation. The mixtures of the protein and XG solutions
(400 mL) were whipped at speed setting 8 (380 rpm) for 20 min. The foams
were immediately transferred to a 20 cm × 20 cm aluminum tray and stored
at -30 ºC for 24 h, followed by freeze-drying for 72 h (FreeZone 18
Liter Console Freeze Dryers, Labconco Corp, Kansas City, MO, USA).

\subsubsection*{Oleogel preparation and
characterization}

{\label{oleogel-preparation-and-characterization}}

\subsubsection*{Preparation of oleogel}

{\label{preparation-of-oleogel}}

Oleogels were prepared by adding a hot mixture (80 \textsuperscript{o}C)
of CO with 0 -- 3\% CW or MAG into 0.5 to 1 g of freeze-dried foams
taken in 50 mL centrifuge tubes. The lipid mixture was prepared by
dissolving the required amount of CW or MAG to CO at 80 ºC. The addition
of oil mixture was stopped when the foam was saturated with the oil, and
the excess oil started coming off when the tubes were inverted. The
tubes were then quickly transferred to a refrigerator
(4\textsuperscript{o}C) to allow the formation of oleogel. The samples
were left in the refrigerator for 24 h prior to any measurements. As a
control, CO containing different concentrations of CW or MAG (without
foam formation) was also added to centrifuge tubes and left in the
refrigerator. Foam-templated oleogel with CO without any CW or MAG was
also kept at the refrigerator as another control.

\subsubsection*{Determination of oil loss from
oleogels}

{\label{determination-of-oil-loss-from-oleogels}}

Oil loss (OL) from the oleogels was used as an indicator of their oil
binding capacity (OBC). OL was determined using a method described by ()
with slight modification. The tubes with oleogels taken out of the
refrigerator were centrifuged for 20 min at 300×g using a tabletop
centrifuge (IEC clinical centrifuge, Damon Corp., Needham Heights, MA,
USA) to remove the excess oil. After centrifugation, the tubes were
inverted and placed on a metal wire mesh, which kept on top of a beaker,
where the released oil was collected. The weight of the tube before and
after removing the released oil was noted. The percent oil loss was
calculated using equation 1,

\(\text{Oil\ Loss}=\frac{W_{a}-W_{b}}{\text{Weight\ of\ the\ oil\ added\ into\ the\ foam}}x\ 100\%\)(1)

where \emph{W\textsubscript{a}} and \emph{W\textsubscript{b}} are the
weights of the tube before and after removing the released oil during
centrifugation, respectively.

\subsubsection*{Microstructure of
oleogels}

{\label{microstructure-of-oleogels}}

The microstructure of the oleogels was obtained using a bright-field
polarized light microscope (Nikon Eclipse E400 microscope, with a Nikon
DS-Fil camera, Nikon Canada Inc., Mississauga, ON) with a 10× objective
lens at room temperature (25 ± 2 ºC). A small amount of freshly-prepared
molten oleogel mixture was placed on a glass slide, covered with a
coverslip, and transferred to a refrigerator for the formation of
oleogels. The microscopy of the oleogels was performed at least after 24
h of storage in the refrigerator.

\subsubsection*{Viscoelasticity of
oleogels}

{\label{viscoelasticity-of-oleogels}}

Viscoelasticity of the oleogels was measured by a rheometer (Model
AR-G2, TA Instruments, Montreal, QC, Canada). A 40mm cross-hatched
parallel plate geometry was used for viscoelasticity analysis to
eliminate any wall-sleep during measurement. Oleogels were gently loaded
on the Peltier plate of the rheometer with a spatula. Excess gel came
out after placing the geometry was gently wiped off to maintain the
correct level. An oscillatory strain sweep (from 0.01 \% to 100 \%) was
applied at a constant frequency of 1 Hz at 25 ºC to find out the linear
viscoelastic region (LVR), and then a frequency sweep measurement was
performed from 0.01 to 100 rad/s at a constant strain of 0.05 \% within
the LVR. The storage (G') and loss modulus (G'') of the samples was
recorded with the TRIOS Software (version 4.0.2.30774, TA Instruments,
Montreal, QC, Canada).

\subsubsection*{Spreadability of Oleogel}

{\label{spreadability-of-oleogel}}

Spreadability of the oleogels was performed using a TA-425 TTC probe
(spreadability-TTC, spreadability RIG) using a texture analyzer (TA-Plus
Texture Analyzer, Texture Technologies Crop. Hamilton, MA, USA) at
compression mode with a penetration depth of 65 mm, test speed of 3 mm/s
and post-test speed of 10 mm/s. Before the measurements, the weight and
distance calibrations were performed. The oleogel sample was placed into
the female cone, pressed gently to avoid incorporation of air using a
plastic spatula, and fixed on the bottom platform of the texture
analyzer. The sample surface was levelled, the male cone was placed at a
defined position (65 mm) above the sample surface, and the experiment
started with the male cone's downward movement (65mm penetration at 3
mm/s speed), which compressed the sample forcing it to flow between the
surfaces of the two cones and return to the initial position. From the
obtained graphic of force vs. distance, the maximum positive force
required for the male cone to penetrate through the sample (firmness)
and the maximum negative force (cohesiveness) were calculated.

\subsubsection*{Preparation and characterization of
cakes}

{\label{preparation-and-characterization-of-cakes}}

\subsubsection*{Cake baking with oleogel}

{\label{cake-baking-with-oleogel}}

Cakes were baked using a slightly modified AACC International Method
10-90.01 (AACC, 1999). In each replicate, batters were prepared using
200 g of all-purpose flour, 280 g of crystalline sugar, 100 g of fat, 24
g of non-fat dried milk, 18 g of dried egg white powder, 6 g of NaCl,
12.5 g of baking powder, and 250 g of water. Mixing of the ingredients
was done using a KitchenAid Ultra Power Mixer (KitchenAid, Whirlpool
Canada LP, Mississauga ON) with a 4.5 qt (4.3 L) stationary bowl and
rotating stirrers. For the fat phase of the batter, either vegetable
shortening, or CO, or FPC and PPC foam-templated oleogels prepared using
3\% MAG and 3\% CW were used. As a control, oleogels prepared using 3\%
CW, 3\% MAG in CO, and freeze-dried foams of PPC-XG and FPC-XG in CO
were also used. Only the 3\% lipid additives were chosen for cake baking
because the best properties of the oleogels were obtained at this
concentration. All the dry ingredients were mixed before adding them
into the mixing bowl. Then the fat phase was added with 150 mL of water
and mixed for 1 min at speed 2 followed by 4 min at speed 4. The rest of
the water was added in steps with various mixing speeds according to the
AACC method. Cakes were baked in an electric oven at 190 ºC (375 °F)
with about 200 grams of dough placed in a baking tray (15.0 × 7.5 × 5.5
cm). After baking, the cakes were cooled at room temperature for 30 min,
carefully removed from the tray, and covered with aluminum foil and
plastic wrap to prevent moisture loss until further analysis.

\subsubsection*{Characterization of cake batters and
cakes}

{\label{characterization-of-cake-batters-and-cakes}}

The microstructure of the batters was obtained using a light microscope
(Nikon Eclipse E400, Nikon Canada Inc., Mississauga, ON) with a 10×
objective lens at room temperature. Batters were analyzed within 30 min
after preparation to minimize the effect of time. A small amount of
batter was taken on a glass slide, compressed gently with a cover slide
and used for image capturing.

The viscosity and viscoelasticity of cake batters were measured using
the AR-G2 rheometer (TA Instruments, Montreal, QC, Canada) with a 40 mm
acrylic parallel plate. Viscosity measurements were done between the two
parallel plates at 25 ºC with a gap of 500 \selectlanguage{greek}µ\selectlanguage{english}m and as a function of
increasing shear rate from 0.01 to 1000 s\textsuperscript{-1}. To find
out the linear viscoelastic region (LVR), G' and G'' were measured at a
constant frequency of 1 Hz at 25 \selectlanguage{ngerman}ºC by controlling the oscillatory
strain sweep from 0.01 \% to 100 \%. Then a frequency sweep measurement
was performed from 0.01 to 100 rad/s at a constant strain of 0.05 \%
within the LVR.

The specific gravity of cake batters was measured as the ratio of the
weight of a certain volume of cake batter to the same volume of water.
The volume of the cakes was determined using rapeseed displacement
procedure according to AACC 10-05 method (AACC, 2001), and the specific
volume of the cakes was measured as the cake volume per unit weight of
the cake (cm\textsuperscript{3}/ g).

The texture profile of the cakes was measured by a two-bite test using a
texture analyzer (TA-Plus texture analyzer, Stable Micro Systems Ltd.
Surrey, UK), according to Kim, Lim, Lee, Hwang and Lee (2017) with
slight modification. The measurements were done 24 h after baking using
cylindrical pieces (2 cm cubes) of cake crumbs. A cylindrical probe of
diameter 2.5 cm, attached to the texture analyzer, was used to compress
the cake pieces two times at a speed of 2 mm/s until the height of the
cake pieces were half. The texture profiles were analyzed using the
Exponent software (version 6.1.4.0, Stable Micro Systems Ltd., Surrey,
UK), according to Friedman, Whitney and Szczesniak (1963) to calculate
the hardness, springiness, chewiness and cohesiveness of the cakes.

\subsection*{Statistical Analysis}

{\label{statistical-analysis}}

All the measurements used in this manuscript were carried out in
triplicate for different foam, oleogel and cake samples, and the results
reported are average and standard deviation of these measurements. The
results were statistically analyzed from the analysis of variance and
t-test at a significance level of 5\% using Microsoft Excel 2013.

\section*{Results and Discussions}

{\label{results-and-discussions}}

\subsection*{Oleogelation using pulse protein stabilized foams in the
presence of CW or
MAG}

{\label{oleogelation-using-pulse-protein-stabilized-foams-in-the-presence-of-cw-or-mag}}

Both FPC-XG and PPC-XG foams readily adsorbed the added canola oil about
30-times their weight with and without CW or MAG and produced a
self-standing solid-like structure after 24 h of storage in a
refrigerator. No oil came out from any oleogels while inverting the
tubes after storing the refrigerator, except the oleogels consists of
only less than 1\% CW or less than 2\% MAG as oleogelator. The critical
concentration (the concentration of oleogelator at which the oleogel
doesn't flow while inverting the tube) of candelilla wax for gelation of
canola oil was previously reported as 1\% (Blake, Co and Marangoni,
2014), which matched with the present study. However, no such report on
the critical concentration of a similar MAG in canola oil at the same
condition was found.

Microstructures of different oleogels (Fig. 1) demonstrated a crystal
network of CW and MAG in the foam-templated oleogels. Oleogels
stabilized only by CW exhibited network of finely dispersed small
grain\selectlanguage{english}-like crystals (Fig. 1m), while MAG oleogel displayed a dense
network of fibril-like crystals (Fig. 1n). Similar microstructures of CW
in CO has been reported before (Blake, Co and Marangoni, 2014). The
microstructure of MAG oleogel was similar to the oleogel prepared using
safflower oil with 4\% MAG (the same ingredient used in the current
study) as reported by Lopez-Martinez, Morales-Rueda, Dibildox-Alvarado,
Charo-Alonso, Marangoni and Toro-Vazquez (2014), but a denser network
and smaller crystals were found in the current study, which might be due
to a different cooling rate and the use of different vegetable oil.

In the foam-templated oleogels with and without CW or MAG, dark rings of
protein foam boundaries were observed under non-polarized light (Fig.
1d-f, and Fig. 1j-l), and crystal network of CW or MAG, similar to that
of CW or MAG-only oleogels, were observed under polarized light (Fig.
1a-c, and Fig. 1g-i). This confirms that the formation of CW or MAG
crystal network formed within the foam-templated oleogels without
altering any protein foam network. Birefringent starch particles with
spherulitic microstructures were also observed in the oleogels
stabilized by pulse protein foams with and without CW or MAG under
polarized light. More starch granules can be seen in FPC oleogels (Fig.
1g-i) compared to PPC oleogels, as the former had more than double
starch content (See Table S1, Supporting Information for proximate
analysis of the pulse protein samples).

\subsection*{Oil loss from the
oleogels}

{\label{oil-loss-from-the-oleogels}}

Oil loss (OL) is an indicator of oil binding capacity (OBC) of oleogel,
higher the OL means lower the OBC. The OL of different oleogels is
displayed in Fig. 2a and 2b. As expected, the OL of both FPC-XG and
PPC-XG foam-templated oleogels significantly reduced with the addition
of both CW and MAG, indicating an increase in OBC of the foam-templated
oleogels with these additives. When there was no additive, the OL was
about 34.4 +- 5.2 \% and 32.4 +- 7.1 \% in the case of PPC-XG and FPC-XG
foam-templated oleogels, respectively. The OL became zero with more than
2\% CW and remained at 4.5 +- 0.2 \% and 3.3 +- 0.5 \% with 3\% MAG in
PPC-XG and FPC-XG foam-templated oleogels, respectively (Fig. 2a). It
should also be noted that CW or MAG alone cannot bind oil as good as in
combination with a protein stabilized foam. The oleogels made only with
1\% or less than 1\% CW or MAG displayed 100\% OL (Fig. 2b), showing
complete disruption of crystal network made of CW or MAG under
centrifugation. No oil was released when 2 \% or more CW was present in
CO; however, about 7.0 +- 0.6 \% oil was released even when 3 \% MAG was
present in the CO. Previously, a significantly higher oil loss was
reported in MAG-stabilized canola oil oleogels, where even with 8\% MAG
about 50 \% of the oil was lost from the oleogel during storage on a
filter paper for 15 days (Lopez-Martinez, Charo-Alonso, Marangoni and
Toro-Vazquez, 2015). This difference could be attributed to the use of
different methods and timescale to measure the OL. Lower OL (higher OBC)
of oleogels containing CW indicates that the crystal network of CW plays
a critical role in determining the OL. Blake, Co and Marangoni (2014)
showed that lower OL could be obtained if the oleogelator can cover more
surface area in the oleogel. Although the dense crystalline network was
found in both CW and MAG systems (Fig. 1), higher OL in MAG oleogels
compared to CW oleogels indicates that needle-shaped MAG crystals (Fig.
1n) formed a weaker network compared to granular and spherulitic CW
crystals (Fig. 1m) in binding CO and preventing disruption during
centrifugation. Although most of the oil was released from the CW or
MAG-only oleogels during centrifugation (with 1\% or below oleogelator),
the presence of protein foam alone significantly reduced OL (Fig. 2a),
which was further improved when the oleogels were stabilized by protein
foam along with CW or MAG.

Intermolecular interactions such as hydrogen bonding, hydrophobic
interactions and van der Waals interactions could be the factors that
helped both CW and MAG crystal network and the foam network to bind
canola oil. In a previous study, it was demonstrated that major
interaction that helped the protein foam network to bind canola oil was
hydrophobic and van der Waals interactions, evidenced by changes in
protein secondary structure and changes in corresponding interaction
bands in FT-IR spectra (Mohanan, 2020). Weak intermolecular hydrogen
bonding was detected in MAG-stabilized oleogels (Lupi, Greco, Baldino,
de Cindio, Fischer and Gabriele, 2016, Ogutcu and Yilmaz, 2014) as well
as in natural wax stabilized oleogels (). The strong crystalline network
formed through intermolecular interactions might have trapped the liquid
oil presented in the protein foam stabilized oleogels, thereby further
reinforcing the overall oleogel network and reducing OL.

\subsection*{Oleogel viscoelasticity}

{\label{oleogel-viscoelasticity}}

Rheological properties of the oleogels are important to understand their
gel strength and better define their application. Oscillatory
strain-dependent viscoelastic measurements of different oleogels were
performed at a constant frequency of 1 Hz to find the linear
viscoelastic region (Fig. S1, Supporting Information). At low strain \%,
the storage moduli (G') of all samples, except 0.5\% CW, and 0.5\% MAG,
were higher than that of loss moduli (G''), indicating an elastic
behaviour. An LVR was observed until about 0.1\% strain. With an
increase in strain, G' decreased, indicating yielding, followed by a
cross over between G' and G'', representing a loss of gel structure.

Frequency-dependent viscoelastic measurements of all oleogels were
performed within the LVR and are shown in Fig. S2 (Supporting
Information). Storage moduli (G') of all samples, except as that of
0.5\% CW, and 0.5\% MAG, were higher than loss moduli (G''), indicating
an elastic behaviour of the gels at the performed range. All oleogels
with CW (Fig. S2a, c, e) displayed similar changes in G' and G'' with
frequency, G' increased linearly with frequency. In contrast, all
oleogels with MAG (Fig. S2b, d, f) displayed a slightly higher rate of
increase in G' at a lower strain, which decreased, making G' more
independent at a higher frequency. Overall, the slope of the G\selectlanguage{english}' curve
was only slightly positive for both CW and MAG, indicating limited
rearrangement of the gel structure at a shorter time scale (or higher
frequency). For all oleogels, G'' decreased first to a minimum and then
increased with frequency (Fig. S2a, c, e), and this behaviour was
evident in oleogels with more than 1\% CW or MAG. The minima in G'' as a
function of frequency indicates motion in the gel network from a more
rigid in-cage structure to a more fluid out-of-cage structure at a
shorter time scale (higher frequency) and weakening of the gel structure
(Zhou, Hollingsworth, Hong, Cheng and Han, 2014). Interestingly, with an
increase in concentration, the minimum values of G'' shifted to a higher
frequency in case of the MAG-based oleogels (Fig. S2b, d, f), while it
shifted to lower frequencies in case of CW-based oleogels (Fig. S2a, c,
e). Similar rheological behaviour was observed for oleogels of vegetable
oil stabilized by sorbitan and glycerol monostearate and is common for
network-based gelled dispersions (Sanchez, Franco, Delgado, Valencia and
Gallegos, 2011).

To compare the gel strength of different oleogels, G' and G'' of
different oleogels at 10 rad/s were replotted in Fig. 3. Both G' and G''
increased with an increase in CW or MAG content, except for FPC
foam-templated oleogels (Fig. 3b), where no change was observed at 2 and
3\% CW or MAG concentrations. Although the viscoelastic moduli values
were much lower for the oleogels without foam, the rate of increase as a
function of CW and MAG concentration was much higher (Fig. 3c) compared
to the foam-templated oleogels. The increase in viscoelastic moduli
could be due to the increase in solid content and the strength of
crystal network with an increase in the concentration of CW or MAG. With
the presence of a protein foam network in the oleogels, the effect of CW
or MAG concentration on gel strength was less visible, especially at 2
and 3\% concentrations. There were no significant differences between
the G' and G'' values when PPC-XG was replaced by FPC-XG
(p\textgreater{}0.05) in foam-templated oleogels at all CW or MAG
concentrations (Fig. 3a, b). In the absence of foam, the oleogels
containing CW displayed higher G' and G'' values than the oleogels made
of MAG at all concentrations (Fig. 3c), which indicates that the CW
crystal network was stronger than that of MAG and are less prone to
deformation. The order of magnitudes of G ' and G'' of 3 \% MAG oleogel
was close to that of 4 \% MAG oleogel created with safflower oil
(Lopez-Martinez, Charo-Alonso, Marangoni and Toro-Vazquez, 2015).
Surprisingly, the foam-templated oleogels containing MAG displayed
higher G' and G'' than the corresponding CW containing oleogels at all
concentrations (Fig. 3a, b). This might be due to the higher oil loss of
MAG containing oleogels (Fig. 2). In our previous study, it was also
demonstrated that when the oil content was lower, the foam-templated
oleogels displayed higher G' and G'' due to the stronger effect of the
protein network on oil gelation (Mohanan, Tang, Nickerson and Ghosh,
2020).

\subsection*{Oleogel spreadability}

{\label{oleogel-spreadability}}

The firmness and cohesiveness of all oleogels were measured using the
spreadability test and are shown in Fig. 4. Firmness and cohesiveness
indicate the easiness of spreading and handling of oleogels; the higher
the firmness, the harder it is to spread the oleogel (lower
spreadability), and higher the cohesiveness, the stickier the oleogel.
As shown in Fig. 4, both the firmness and cohesiveness of all oleogels
increased with an increase in CW and MAG concentrations as expected.
When there was no foam present (Fig. 4c and 4d), The firmness and
cohesiveness of the oleogels were much lower compared to the
foam-templated oleogels (Fig. 4a and 4b). Between these two oleogels
without foam, CW oleogels displayed higher firmness and cohesiveness
than MAG oleogels (Fig. 4c and 4d). This agrees with the rheology
results, where the G\selectlanguage{english}' of CW oleogels was significantly higher than that
of MAG oleogels. Therefore, it is reasonable to assume that the firmness
of oleogels is associated with the strength of the crystal network and
its ability to retain oil. In both FPC-XG and PPC-XG foam-templated
oleogels, however, there was no significant difference in firmness or
cohesiveness when CW was replaced by MAG (p\textgreater{}0.05) at all
concentrations, except when the cohesiveness decreased at 3\%
oleogelator concentration. Nonetheless, unlike what has been observed in
rheological properties, FPC-XG foam-templated oleogels displayed higher
firmness and cohesiveness than corresponding PPC-XG foam-templated
oleogels with and without CW or MAG.

\subsection*{Cake baking with oleogel}

{\label{cake-baking-with-oleogel-1}}

Conventionally, a highly saturated fat containing shortening is used in
cake baking to stabilize air bubbles in the batter and to provide the
right rheology of the batter to give a soft tendered final cake product
(Wilderjans, Luyts, Brijs and Delcour, 2013). In the present work,
shortening was fully replaced with oleogels to determine how much of
these oleogels can mimic the functionality of shortening. To do this, we
used PPC-XG and FPC-XG foam-templated oleogels with and without 3\% CW
and 3\% MAG, and oleogels made of 3\% MAG and 3\% CW alone. As a
control, the cake was also baked using 100\% CO as a control. Note that
the oleogels used in cake baking contained more than 95\% CO.

\subsubsection*{Properties of cake
batters}

{\label{properties-of-cake-batters}}

The specific gravities of the cake batters prepared using vegetable
shortening, CO and the various oleogels are shown in Table 1, and their
microstructures obtained using a light microscope are shown in Fig. 5.
The batter prepared using shortening displayed the least specific
gravity, indicating higher incorporation of air into the batter
(Wilderjans, Luyts, Brijs and Delcour, 2013). This is also evident in
their microscopic image, where numerous small air bubbles can be
observed (Fig. 5). The conventional shortening used in the research
consists of solid fat crystals and mono and diglyceride emulsifiers,
which are capable of stabilizing air bubbles in the batter (Wootton,
Howard, Martin, McOsker and Holme, 1967). Batter prepared using CO
displayed the highest specific gravity, which could be due to the
inability of the liquid oil to stabilize air bubbles in the batter. As
illustrated in Fig. 5, there were only a few air bubbles incorporated in
CO batter. Oleogels consist of only foams or CW or CW with foam
displayed similar but significantly higher specific gravity compared to
shortening. The microscopic images of these batters shown in Fig. 5 also
confirmed the incorporation of fewer air bubbles in the compared to
shortening batter. Among all the oleogels, those consist of MAG with or
without foam displayed lowest specific gravity; however, they were still
higher than that of shortening batter (Table 1). The oleogels consist of
MAG and protein foams displayed similar specific gravity as that of
batters with only MAG oleogel (p \textgreater{} 0.05), indicating the
presence of the protein foams did not help further air incorporation in
the batter. Microscopic images of batters containing MAG clearly showed
the incorporation of a greater number of air bubbles in the batter
compared to CO, only protein foams and CW batters (Fig. 5). It also
shows that the presence of solid fat crystals made of MAG was crucial
for the stabilization of air in the batter, which could be ascribed to
the surface activity of the MAG. Although CW was able to form crystals
in CO, the cake batters prepared using only CW displayed almost similar
specific gravity as that of CO batter (p \textgreater{} 0.05). The
addition of protein foams in the oleogels with CW decreased the specific
gravity, but it not significantly different than the batters prepared
using only protein foam-templated oleogels. These results show that not
only improvement on gel strength or OBC of oleogels can improve the
quality of batters, but the ability of the oleogelators to increase the
stabilization of air bubbles does matter.

It is known that the rheological properties of the batters determine the
functional properties of the final product (Ronda, Oliete, Gomez,
Caballero and Pando, 2011). Therefore, both flow and viscoelastic
behaviour of all batters were determined (Fig. 6). All cake batters
displayed pseudoplastic behaviour, where viscosity decreased with an
increase in shear rate (Fig. 6a). The flow curves of all cake batters
were fitted to a power-law model (\(n=K{\dot{\gamma}}^{n-1}\)), where \selectlanguage{english}n is the
viscosity, \(\dot{(\gamma)}\) is the shear rate, K is the consistency
coefficient, and n is the flow behaviour index. The fitting parameters
are shown in Table 2 (R\textsuperscript{2} values were \textgreater{}
0.83 for all). Flow behaviour indices of all batters were less than 1,
and shortening batter displayed the lowest flow behaviour index followed
by the oleogel batters consisting of both foam and MAG, indicating more
shear thinning and the formation of higher-order structure. Batters with
both foam and CW displayed the highest `n' values (Table 2), indicating
less shear thinning behaviour. Moreover, the shortening batter displayed
the highest consistency index (K), indicating higher viscosity at 1
s\textsuperscript{-1} shear rate than that of all other batters (Fig.
6a). The higher low-shear viscosity of shortening batter might be
attributed to solid fat crystals and higher air incorporation. At higher
shear rates, the viscosities of all batters were close to each other,
where the fat crystal network present in the shortening could be
disrupted and the air bubbles collapsed, leading to a reduction in
viscosity. Due to the inclusion of a higher portion of liquid oil in the
oleogel and CO batters, they displayed lower viscosities than the
shortening batter. Reduction in viscosity while replacing shortening
with rapeseed oil was previously reported (Hesso, Garnier, Loisel,
Chevallier, Bouchet and Le-Bail, 2015). The n and K values of the PPC
and FPC foam-templated oleogel cake batters were also comparable to that
of muffin batters prepared using HPMC foam-templated oleogel batters
(Lee, 2018), indicating similar functionality of protein and HPMC foam
in oleogels.

All batters displayed tan \selectlanguage{greek}δ \selectlanguage{english}(ratio of G'' to G') values lower than 1
(Fig. 6b) and higher G' than G'' at the whole frequency range studied
(Fig. 6c and 6d), indicating elastic behaviour. Tan \selectlanguage{greek}δ \selectlanguage{english}for shortening
batter decreased with an increase in frequency, indicating dominant
elastic behaviour. All batters displayed similar tan \selectlanguage{greek}δ \selectlanguage{english}to the shortening
batter below 1 rad/s frequency, however, it raised beyond 1 rad/s and
exceeded the value of shortening batter, indicating replacement of
shortening with oleogels led to more contribution to the viscous nature
of the batter. Both G' and G'' also increased linearly with increase in
frequency, indicating structural changes with the lowering of the
timescale of the applied strain. The increase in G' with frequency was
less in shortening batters compared to the others. When shortening was
replaced with oleogels or CO, both G' and G'' reduced significantly. The
batters prepared using wax-based oleogels (Lim et al., 2017, Kim et al.,
2017) and HPMC foam-templated oleogel (Lee, 2018) also displayed similar
viscoelastic behaviour as that of the batters used in the current study.
Both G' and G'' increased with increase in frequency when shortening was
replaced with HPMC foam-templated oleogels (Lee, 2018). Lower viscosity
and viscoelastic values of oleogel batters compared to shortening batter
could be related to partial replacement of solid fat by liquid oil and
lower air incorporation. An increase in specific gravity due to lower
air incorporation was led to a reduction in cake volume and an increase
in cake hardness (Sahin, 2008). The ability of the batter to retain the
air determines their rheological properties and the quality of the final
product.

\subsubsection*{Properties of cakes}

{\label{properties-of-cakes}}

The cross-sectional view of the final cake products and their specific
volumes (cm\textsuperscript{3}/g) are provided in Fig. 7 and Table 1,
respectively. Shortening cakes displayed the uniform distribution of
open air cells and granular particles of flour solids, while cakes
prepared using CO and oleogel displayed the relatively non-uniform
distribution of large air bubbles dispersed in a dense network of cake
matrix. Oleogels prepared using both the foams with and without CW or
MAG displayed similar cakes appearance. However, contradictory to what
has been observed in the literature (Sowmya, Jeyarani, Jyotsna and
Indrani, 2009), there was no volume reduction observed when shortening
was replaced by liquid oil or oleogel, all cakes displayed similar
specific volume (p\textgreater{}0.05). These results confirm that the
rheology or specific gravity of the batters cannot be used to predict
the final cake volume. There might be interactions with other
ingredients during baking, which determined the volume of the final
product.

Textural properties of cakes (hardness, springiness, cohesiveness, and
chewiness) are displayed in Fig. 8. Hardness is an indicator of staling
of baked products and can be determined using the force required to
compress the sample to a certain height. The shortening cakes displayed
the lowest hardness (p \textless{} 0.05), followed by the hardness of
cakes prepared by PPC and FPC foam-templated oleogels containing MAG,
which were lowest among all the oleogel and CO-added cakes (Fig. 8a).
Cakes prepared using CO and CW oleogel displayed similar, but highest
hardness among all the cakes where shortening was replaced with another
lipid. All other oleogel cakes (MAG, PPC-XG, FPC-XG, PPC-XG+CW, and
FPC-XG-CW) displayed similar hardness. The hardness of the cakes
correlated well with their batter specific gravity. Batters with lower
specific gravity displayed lower hardness, and these results are
supported by the literature (Sahin, 2008). Higher hardness also
indicates higher protein-protein and protein-gelatinized starch
interaction (Paraskevopoulou and Kiosseoglou, 1997). The
monoacylglycerols/diacylglycerols present in the shortening interacted
with starch and reduced its interaction with protein and hence lowering
of the staling process (Cauvain, 1998, Mattil, 1964), which led to the
lowest hardness of shortening cakes and lower hardness of MAG-based
oleogel cakes. In addition to this, shortening saturated fat crystals'
ability to prevent the formation of gluten network might have also led
to lower hardness. Generally, springiness values (Fig. 8b) were found to
be very close to each other, although CO cake displayed slightly higher,
and MAG oleogel cake displayed slightly lower springiness than
shortening cake (p \textless{} 0.05). Springiness is an indication of
the elasticity of cake, and our results showed that all the cakes had
almost similar capacity to recover its deformation after exposed to any
external force. Cohesiveness is a direct function of work required to
breakdown the internal bonds between different ingredients in the cake
matrix during each chew or the internal resistance of food to traction,
which was determined from the ratio of the second to the first peak area
of the two-bite test. Shortening cakes displayed the highest
cohesiveness followed by all CO and all oleogels cakes, except the cakes
made of oleogels consist of CW, which displayed the lowest cohesiveness
(Fig. 8c). This indicates that the replacement of shortening with liquid
oil and oleogels make it easy to disintegrate the cake matrix during
mastication. Both springiness and cohesiveness did not correlate well
with any batter characteristics studied, such as viscosity,
viscoelasticity or specific gravity. Therefore, springiness and
cohesiveness of cakes could be influenced by the changes in the
interaction between the ingredients and how they form the structure in
the cake matrix during baking. The chewiness of the cakes followed a
similar trend as that of hardness, except the CW oleogel cake, which
displayed lower chewiness than CO cakes (Fig. 8d). Chewiness indicates
the energy required for mastication, which was calculated from the
product of primary parameters hardness, cohesiveness and elasticity
(Friedman, Whitney and Szczesniak, 1963). Chewiness values indicate that
the CO cakes required the highest energy to masticate, and the
replacement of CO with all oleogels decreased the chewiness to a level
similar to shortening-based cake.

\section*{Conclusions}

{\label{conclusions}}

The effect of small fractions of high melting candelilla wax (CW) and
monoacylglycerols (MAG) in pea (PPC) or faba bean protein concentrate
(FPC) plus xanthan gum (XG) foam-templated oleogels on oil loss,
rheological properties, spreadability and functionality as a shortening
alternative in cakes were examined. The study showed that the addition
of 3\% CW and MAG in foam-templated oleogels could prevent or
significantly reduce oil loss, and increase storage and loss modulus
\textasciitilde{} 1000 times, and firmness and cohesiveness by
\textasciitilde{} 3 times. Although CW was more effective in reducing
the OL, and increasing the rheological properties, MAG was more
effective in improving the functional properties of cake batters and
cakes. Even though it was not as good as shortening baked cakes in terms
of hardness, cohesiveness and chewiness, hardness and chewiness of
foam-templated oleogel cakes were significantly reduced with the
incorporation of MAG into the oleogel, rather than using liquid canola
oil.

\section*{Acknowledgements}

{\label{acknowledgements}}

This work was supported by the Agriculture Development Fund (ADF) from
the Saskatchewan Ministry of Agriculture and the Canada -- Saskatchewan
Growing Forward 2 bi-lateral agreement and, Saskatchewan Pulse Growers
(project\# 20160136); CFI-John R. Evans Leaders Fund (2012-31292), and
Innovation and Science Fund (ISF) from Saskatchewan Ministry of Advanced
Education. Technical help from the Grains Innovation Lab, University of
Saskatchewan, is also acknowledged.

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