Experimental Procedures
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
~70 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).
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).
Oleogel preparation and
characterization
Preparation of oleogel
Oleogels were prepared by adding a hot mixture (80 oC)
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 (4oC) 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.
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 Wa and Wb are the
weights of the tube before and after removing the released oil during
centrifugation, respectively.
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.
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).
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.
Preparation and characterization of
cakes
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.
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 µm and as a function of
increasing shear rate from 0.01 to 1000 s-1. To find
out the linear viscoelastic region (LVR), G’ and G” were measured at a
constant frequency of 1 Hz at 25 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
(cm3/ 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.
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.