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.