*2.1. Reagents and Materials*

Chickpeas (*Cicer arietinum* L.), mustard, salt, vinegar, sugar, nutritional yeast, and eggs were purchased in a local market. Refined rapeseed oil (RRO), cold-pressed rapeseed oil from *Brassica napus* L. (CPRO), cold-pressed sunflower oil from *Helianthus* L. (CPSO), cold-pressed linseed oil from *Linum usitatissimum* L. (CPLO) and cold-pressed camelina oil from *Camelina sativa* L. (CPCO) were kindly provided by a local vegetable oil factory in the original packing (1 L of RRO in polyethylene terephthalate (PET) bottle, 250 mL of cold-pressed oils in amber-colored glass bottles, marasca type).

Additionally, three mayonnaises (MT1, MT2—traditional recipe mayonnaises (full-fat, 76 and 68%, respectively, and MV—vegan mayonnaise containing 35% fat) packed in colorless glass jars were supplied directly after production by two different manufacturers representing the top-selling brands in the Polish market.

All oil and mayonnaise samples were within their stated shelf lives and stored in a refrigerator at 4 ◦C until analysis (no longer than 4 days after opening the original packaging).

All chemicals of analytical grade were purchased from Sigma-Aldrich (Pozna ´n, Poland). Redistilled water was used for the preparation of solutions.

### *2.2. Preparation of Mayonnaise Samples*

Aquafaba, the liquid from the chickpea jars, was separated using a stainless-steel mesh kitchen strainer. A representative sample of aquafaba was taken for analysis and mayonnaise preparation. The content of each ingredient for the preparation of mayonnaise samples was selected based on preliminary tests. Four mayonnaise batches (MRO, MSO, MLO and MCO) of 200 g were prepared according to the procedure described by Raikos et al. [23] with some modifications using the ingredients listed in Table 1. Aquafaba was mixed with mustard, vinegar, nutritional yeast, salt, and sugar using a high mixing bowl and a stick blender (BOSH® MSM67160, Robert Bosch GmbH, Gerlingen, Germany). Then, blends of RRO with each cold-pressed oil were gradually and slowly added to the aqueous mixture during the blending procedure to achieve the proper consistency. Emulsions were homogenized for 5 min utilizing a homogenizer (DT basic, Yellow Line, IKA®-Werke GmbH & Co., KG, Staufen, Germany) at 8000 rpm. The appearance of the prepared aquafaba-based emulsions is shown in Figure 1. Mayonnaises were packaged into colorless glass jars and stored at 4 ◦C in a refrigerator until analyses (no longer than 4 days).


**Table 1.** Ingredients of different formulations of vegan mayonnaise samples.

MRO—mayonnaise with a blend of refined rapeseed oil (RRO) and cold-pressed rapeseed oil (CPRO); MSO— mayonnaise with a blend of refined rapeseed oil (RRO) and cold-pressed sunflower oil (CPSO); MLO—mayonnaise with a blend of refined rapeseed oil (RRO) and cold-pressed linseed oil (CPLO); MCO—mayonnaise with a blend of refined rapeseed oil (RRO) and cold-pressed camelina oil (CPCO).

**Figure 1.** The appearance of the prepared aquafaba-based mayonnaise samples with blends of refined rapeseed oil and cold-pressed rapeseed oil (MRO), cold-pressed sunflower oil (MSO), cold-pressed linseed oil (MLO), and cold-pressed camelina oil (MCO).

### *2.3. Characterization of Mayonnaise Ingredients*

2.3.1. Determination of Emulsifying Properties

The emulsifying activity index (EAI) and the emulsion stability index (ESI) were determined according to the procedure described by Cheung et al. [31] but developed initially by Pearce and Kinsella [32]. In brief, 5.0 g of aquafaba or egg yolk was homogenized with 5.0 g of RRO using a homogenizer at a speed of 8000 rpm for 5 min. Then, a 50 μL aliquot of the emulsion was diluted to 7.5 mL of 0.1% sodium dodecyl sulphate (SDS) and vortexed using a classic vortex mixer (Velp Scientifica Srl, Usmate (MB), Italy) for 10 s. The absorbance of the diluted emulsion samples was measured at λ = 500 nm by a Hitachi U-2900 spectrophotometer (Tokyo, Japan). The EAI and ESI were calculated using the following equations:

$$\text{EAI} \left( \frac{\text{m}^2}{\text{g}} \right) = \frac{2 \cdot 2.303 \cdot A\_0 \cdot N}{c \cdot \text{g} \cdot \phi \cdot 10000} \tag{1}$$

$$\text{ESI} \left(\text{min}\right) = \frac{A\_0}{A\_0 - A\_{10}} \cdot t \tag{2}$$

where, *A*0 and *A*10 are the absorbance values measured at an initial time, and after 10 min, respectively, *t* is the time interval (10 min), *N* is the dilution factor, *c* is the protein concentration (g/mL), *ϕ* is the oil volume fraction of the emulsion and *φ* is an optical path (1 cm).

### 2.3.2. Determination of Protein Concentration

The protein concentration was determined with the Kjeldahl method, according to the official Polish Standard Method PN-75/A-04018 [33]. The protein concentration was estimated as the nitrogen content multiplied by a conversion factor of 6.25.

### 2.3.3. Determination of Fatty Acid Composition

Fatty acid profiles for RRO and all cold-pressed vegetable oils (CPRO, CPSO, CPLO and CPCO) were determined in accordance with the official method ISO 5508 [34]. Fatty acid methyl esters (FAMEs) were prepared by the transesterification of oil samples carried out in methanol using potassium hydroxide as a base and the derivatization of fatty acids was conducted following the procedure described by ISO 5509 [35].

The quantification of fatty acids was performed by applying a gas chromatograph (HP 5890 GC) equipped with a flame-ionization detector (FID) (Hewlett-Packard, Avondale, PA, USA) and a high polar capillary column BPX 70 (60 m × 0.25 mm, 0.25 μm). The temperatures of the injector and detector were adjusted to 250 ◦C, while the oven temperature program was as follows: heating from 150 to 210 ◦C at 1.3 ◦C/min and holding at 210 ◦C for 5 min. The carrier gas was helium at a flow rate of 0.6 mL/min.

The identification of fatty acids was accomplished using external FAME standards, and the results are presented as weight percentages of total fatty acids.

### 2.3.4. Determination of Radical Scavenging Activity

The RSA values of aquafaba, oils (RRO, CPRO, CPSO, CPLO and CPCO), mustard and nutritional yeas<sup>t</sup> were analyzed spectrophotometrically by DPPH and ABTS methods according to the modified procedures previously described in detail [36].

The liquid aquafaba sample was dissolved in methanol and used directly for RSA measurements.

The test tubes with oils (3.00 g), mustard (2.00 g) and nutritional yeas<sup>t</sup> (0.50 g) with methanol (5 mL) were shaken for 30 min using a shaker SK-0 330-PRO (CHEMLAND, Stargard Szczeci ´nski, Poland) at room temperature. The extracts were separated from oils in a freezer ( −20 ◦C, 30 min) and transferred quantitatively into glass bottles. Each studied sample was extracted in triplicate, and extracts were stored in a refrigerator prior to RSA analysis.

In the case of the DPPH test, 0.3 mL of 0.1% methanolic solution of aquafaba (*v*/*v*) or 0.2–0.5 mL of the methanolic extracts of oils, mustard and nutritional yeas<sup>t</sup> were added to 1.8–1.5 methanol and 0.5 mL of DPPH methanolic solution (304 μmol/L). The mixtures were shaken vigorously and left in darkness for 15 min. The absorbance of each sample was measured at 517 nm against a reagen<sup>t</sup> blank (2 mL of methanol and 0.5 mL of DPPH methanolic solution) using a Hitachi U-2900 spectrophotometer in a 1 cm glass cell.

For the ABTS assay, 0.1 mL of 0.1% methanolic solution of aquafaba (*v*/*v*) or 0.1–0.3 mL of the methanolic extracts of oils, mustard and nutritional yeas<sup>t</sup> were added to 2.4–2.2 mL of ABTS•<sup>+</sup> reagen<sup>t</sup> (diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm). The obtained mixtures were incubated at 30 ◦C for 1 min and the absorbance of each sample was measured at 734 nm against a reagen<sup>t</sup> blank (2.5 mL of ABTS•<sup>+</sup> solution).

The RSA values were expressed as micromoles of Trolox equivalents (TE) per 100 g of the studied sample.

### *2.4. Characterization of Mayonnaise Samples*

### 2.4.1. Determination of Radical Scavenging Activity

The QUENCHER-DPPH and QUENCHER-ABTS extraction-free procedures were applied for a direct evaluation of the RSA of the proposed vegan and commercial mayonnaises.

For the QUENCHER-DPPH assay, 0.0300–0.0500 g of the mayonnaise samples was transferred into centrifuge tubes. The reaction was started by adding 6 mL of DPPH solution (60.8 μmol/L). The mixture was vortexed for 5 min and left in darkness for 15 min. The samples were centrifuged at 3120× g (centrifuge MPW-54, MPW MED. INSTRUMENTS, Warsaw, Poland) for 3 min and the absorbance of optically clear supernatant was measured at 517 nm.

For the QUENCHER-ABTS assay, 0.0200–0.0300 g of the mayonnaise samples was weighed to centrifuge tubes and the reaction was started by adding 6 mL of ABTS•<sup>+</sup> reagen<sup>t</sup> (diluted with ethanol to an absorbance of 0.70 ± 0.02 at 734 nm). The mixtures

were vortexed for 5 min and centrifuged at 3120× *g* for 3 min. The absorbance of clear supernatants was measured at 734 nm against a reagen<sup>t</sup> blank (2.5 mL of ABTS•<sup>+</sup> solution).

### 2.4.2. Determination of Oxidative Stability

The lipid phase of each mayonnaise sample was separated according to the procedure [37] for an analysis of oxidative stability. Mayonnaises were frozen at −18 ◦C for 12 h and then thawed for 12 h at 4 ◦C to break the emulsion. The mixture was centrifuged for 5 min. Each lipid phase, separated from the emulsion residue, was stored in a closed glass flask in the refrigerator prior to further analysis.

The peroxide value (PV), anisidine value (AnV) and acid value (AV) of the lipid phases were determined to estimate the formation of primary and secondary oxidation products as well as free fatty acids, respectively, that affect the rancidity and the mayonnaise stability.

The PV was measured by iodometric titration according to the official procedure ISO 3960:2017 [38] and was expressed as milliequivalents of active oxygen per kilogram of lipid phase (mEq O2/kg).

The AnV was analyzed according to the ISO 6885:2016 method [39].

The oxidation state of the lipid phase given by the TOTOX index was calculated according to the formula: (TOTOX = 2PV + AnV).

However, the AV was analyzed according to the ISO 660 (1996) method [40].
