# Air-water interfacial and foaming properties of crude and purified globulins from pigeon pea seeds

Adilson Roberto Locali-Pereira, Joëlle Davy, Eléna Keuleyan, Véronique Solé-Jamault, Valérie Beaumal, Renata Tieko Nassu, Rodolfo Godoy, Vânia Regina Nicoletti, Claire Berton-Carabin, Adeline Boire

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## To cite this version:

Adilson Roberto Locali-Pereira, Joëlle Davy, Eléna Keuleyan, Véronique Solé-Jamault, Valérie Beaumal, et al.. Air-water interfacial and foaming properties of crude and purified globulins from pigeon pea seeds. Food Hydrocolloids, 2026, 172 (3), pp.112131. 10.1016/j.foodhyd.2025.112131. hal-05499953

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# Air-water interfacial and foaming properties of crude and purified globulins from pigeon pea seeds

Adilson Roberto Locali-Pereira<sup>a</sup>, Joëlle Davy<sup>b</sup>, Eléna Keuleyan<sup>b</sup>, Véronique Solé-Jamault<sup>b,D</sup>, Valérie Beaumal<sup>b</sup>, Renata Tieko Nassu<sup>c</sup>, Rodolfo Godoy<sup>c</sup>, Vânia Regina Nicoletti<sup>a,D</sup>, Claire Berton-Carabin<sup>b,d,*</sup>, Adeline Boire<sup>b</sup>

## ARTICLE INFO

**Keywords:**

Plant proteins

Crude protein

7S globulin

Interfacial rheology

Foam

Foamability

## ABSTRACT

Protein concentrates or isolates are widely used by the food industry to produce aerated foods. Although the foamability is primarily attributed to proteins, highly surface-active minor compounds are also present in these ingredients, and their presence must be considered when investigating their air-water interfacial properties. In this study, the air-water interfacial and foaming properties of crude and purified globulin extracts from pigeon pea seeds were evaluated. At low bulk concentration, no difference in the interfacial behavior was observed between crude and purified globulins. At high bulk concentration, crude globulin extracts formed less rigid interfacial films compared to purified globulins, with the latter leading to viscoelastic solid-like interfacial layers. This suggests that in more concentrated suspensions, minor compounds of the crude extracts accumulate more at the interface, competing with globulins for adsorption, which leads to a reduction in the mechanical rigidity of the interfacial film. Purified globulins demonstrated superior aqueous foam stability compared to whey protein isolate and bovine serum albumin, highlighting the potential of pigeon pea globulins as effective foaming agents in food applications.

## 1. Introduction

Proteins are foaming agents commonly used in food systems to produce aerated products such as mousses, ice creams, cakes and beverages (Damodaran, 2006; Narsimhan & Xiang, 2018; Zhu et al., 2020). Proteins of animal origin, such as those present in egg white or milk, are the most used by the food industry due to their well-known good performance in the formation and stabilization of foams (Shen et al., 2025; Völp et al., 2021; Yang et al., 2023). However, in recent years, the use of plant proteins in these products has grown significantly, driven by their environmental benefits, sustainability, and broad availability (Guo et al., 2025). With the growing incentive to partially shift from the use of animal proteins to plant proteins, several studies have investigated alternative sources of foaming agents based on proteins from different seeds, from the most conventional, like soybean and pea, to less widely encountered ones, such as mung bean, Bambara bean, lupin, cowpea and

others (Rangel et al., 2003; Völp et al., 2021; Vondel et al., 2023; Yang et al., 2022a,b, 2023). The degree of purity and the use of mild processes to obtain plant proteins in their most native state have shown to be critical factors for good interfacial and foaming properties (Barozzi et al., 2025; Yang & Sagis, 2021).

Pigeon pea (Cajanus cajan L. Millsp.) is an unconventional pulse native to countries in Asia and Africa, with a protein content of around 20 wt% (Fernández Sosa et al., 2021; Sun et al., 2020). Globulins are the main proteins present, belonging to the 7S globulin fraction (Krishnan et al., 2017; Singh et al., 1990). Conventional protein extraction methods have been used to produce pigeon pea globulin extracts to evaluate their functional properties (Adenekan et al., 2018; Fernández Sosa et al., 2021, 2023; Mwasaru et al., 1999) Such methods consist of the solubilization of proteins in alkaline conditions (pH 8.0–12.0) followed by isoelectric precipitation (pH 4.0–5.0), allowing the recovery of protein extracts rich in globulins (Gravel & Doyen, 2023). Although such

<footnote><sup>a</sup> Unesp - São Paulo State University, Institute of Biosciences, Humanities and Exact Sciences, Department of Food Engineering and Technology, Cristóvão Colombo Street, 2265, 15054-000, São José do Rio Preto, SP, Brazil</footnote>

<footnote><sup>b</sup> INRAE, UR BIA, F-44316, Nantes, France</footnote>

<footnote><sup>c</sup> Embrapa Southeastern Livestock - Brazilian Agricultural Research Corporation (Embrapa), São Carlos, Brazil</footnote>

<footnote><sup>d</sup> Laboratory of Food Process Engineering, Wageningen University & Research, P.O. Box 17, 6700 AA, Wageningen, the Netherlands</footnote>

<footnote>* Corresponding author. INRAE, UR BIA, F-44316, Nantes, France
E-mail address: clair.e.carabin-berton@wur.nl (C. Berton-Carabin).</footnote>
methods allow the recovery of a high amount of proteins, protein precipitation can cause modifications in their native structures, compromising their functionality (Kumar et al., 2021). Thus, milder aqueous fractionation techniques, without protein precipitation step, enable the recovery of proteins in their most native state possible. Yet, a potential drawback is that substantial contents in non-protein compounds such as sugars, starches, phospholipids and phenols may be present in protein extracts obtained by these methods, with possible impacts on their functionality (Yang et al., 2022a,b, 2023).

The purification of globulins allows the reduction of these interferents, based on the isolation of the target protein (Gravel & Doyen, 2023). By using pure proteins in studies of functional properties, it is possible to infer their individual behavior and, consequently, better understand their role in the overall functionality of protein extracts, which generally contain other groups of proteins and minor compounds. Krishna and Bhatia (1985) were pioneers in proposing the purification of pigeon pea 7S globulin, using chromatography techniques. The authors obtained native 7S globulins with a molecular weight of 180 kDa and two types of subunits with molecular weights of 72 and 57 kDa. However, due to the difficulty of purifying proteins in sufficient quantities, the authors did not investigate their functional properties. For this, purification on a preparative scale is necessary to obtain purified fractions on the gram scale (Gueguen et al., 1984). In previous work by our research group (Locali-Pereira et al., 2025), a protocol was developed for the purification of 7S globulin from pigeon pea on a large scale, which allows obtaining these fractions in sufficient quantities to probe these properties.

To the best of our knowledge, no study in the literature has evaluated the foaming properties of pigeon pea 7S globulin, but some studies using protein concentrates or isolates thereof have been reported (Adenekan et al., 2018; Akintayo et al., 1999; Mwasaru et al., 1999). It has been demonstrated that surface-active minor compounds present in these protein-rich ingredients, such as phospholipids or saponins, also adsorb at the air-water interface and can interfere with the functional properties of proteins (Bock et al., 2021; Grasberger et al., 2024; Keuleyan et al., 2024). Phospholipids, classified as polar lipids, can exhibit strong interfacial activity, enabling them to compete with proteins for adsorption at the air-water interface. This competition may lead to the formation of heterogeneous interfacial films or result in the displacement of proteins from the interface, resulting in poor foaming properties (Yang, Berton-Carabin, et al., 2022; Dickinson 2011). On the other hand, protein complexation with phenolic compounds and saponins has been shown to improve foaming properties. Anthocyanins at concentrations ranging from 0.05 to 0.2 % w w⁻¹, covalently complexed with soy proteins, improved their foaming properties (Sui et al., 2018). The same was observed for pea protein isolate complexed with tea saponin (Xie et al., 2023). The use of purified protein fractions as standards, compared to protein fractions of lower purity, allows for the effective characterization of the intrinsic properties of the proteins and the respective role of other compounds at the air-water interface. The availability of an established mild purification protocol for the globulin fraction of pigeon pea (Locali-Pereira et al., 2025) enables the preparation of both crude and partially purified protein extracts from the same raw material. This facilitates a controlled comparison of how compositional complexity influences interfacial and foaming properties. Therefore, pigeon pea serves both as a model for investigating underutilized protein sources and as a practical system for evaluating the impact of minor constituents on protein functionality.

In this context, understanding how proteins from different extract types behave at fluid interfaces is essential to elucidate the mechanisms behind their functional performance. When adsorbed at the interface, protein chains rearrange and can unfold to a certain extent to form the interfacial film (Narsimhan & Xiang, 2018; Wierenga & Gruppen, 2010). The behavior of proteins at the air-water interface can be investigated by measuring the rheological properties of the interfacial film, allowing inferences on the mechanical properties and the microstructure of the

layer formed. In addition, studying adsorption dynamics – such as by monitoring the time evolution of interfacial tension or transient interfacial rheological measurements – provides valuable insights into how proteins adsorb, rearrange, and contribute to the development of interfacial structure and viscoelasticity over time (Dickinson, 2011).

In this work, the air-water interfacial and foaming properties of three different ingredients based on pigeon pea were evaluated: (i) crude protein extract obtained from commercial seeds (cv. unknown) (CPE-C); (ii) crude protein extract obtained from pure line seeds (cv. g18-95) (CPE-E); and (iii) purified 7S globulin (7S). The adsorption of constituents in these samples and the rheological behavior of the films formed at the air-water interface were investigated, as well as foamability and foam stability. Whey protein isolate (WPI) and bovine serum albumin (BSA) were used as control for good foaming agents, while lysozyme (LYZ) was used as a control for poor foaming agent. The findings of this study demonstrate the potential of pigeon pea proteins as foaming agents and reveal that at low bulk concentrations, non-protein compounds in these ingredients have a greater impact on air-water interfacial properties.

## 2. Materials and methods

### 2.1. Materials

Pigeon pea seeds were purchased from a local supplier (cv. unknown) (São Paulo, Brazil) and donated (cv. g18-95) by the Brazilian Agricultural Research Corporation (EMBRAPA). Sodium phosphate (≥99 %) was purchased from Sigma-Aldrich (Missouri, USA). Protein assay dye reagent and Coomassie blue were purchased from Bio-Rad (California, USA). Tris-glycine SDS running buffer and Tris base (≥99.8 %) reagent were purchased from ThermoFisher (Massachusetts, USA). Bovine serum albumin was supplied by MP Biomedicals (California, USA). Whey protein isolate (Prolacta®95) was supplied by Laczalis BBA (Rétiers, France). Lysozyme was supplied by OVONOR (Tregueux, France).

### 2.2. Crude protein extraction

Two different types of seeds were used to extract the proteins: commercial seeds (cv. unknown) and pure line seeds (cv. g18-95). The seeds were ground to produce flour (<300 µm) using a disk mill (J.R. Araújo & Cia Ltda., Brazil) at 1730 rpm. For protein extraction, 200 g of flour were dispersed in 1 L of 50 mM Tris buffer solution pH 8.0. The suspension was stirred for 1 h in a mechanical stirrer (RZR 2041; Heidolph, Baviera, Germany) at 225 rpm. After stirring, the suspension was centrifuged (10000 g × 10 min × 4 °C) and the supernatant was collected. Another 1 L of 50 mM pH 8.0 Tris buffer solution was added to the pellet followed by agitation and centrifugation with collection of the supernatant. The two supernatants were pooled, centrifuged again in the same conditions, and filtered (8-µm MCE membrane, Millipore) to remove small particles. To remove the salts and small molecules like polyphenols, the extracts were injected into a desalting column filled with 7.5 L of Matrix celluline (GH25 cellulose gel, 45–105 µm Amicon-Millipore). The column was equilibrated with pH 8.0 sodium carbonate buffer. The injection was conducted at a flow rate of 50 mL/min. The collected volume was 2.8 L for each extract. The desalted extracts were frozen and freeze-dried for 72 h. It was previously shown that this desalting step does not result in the removal of protein compounds (Locali-Pereira et al., 2025). The crude protein extracts (CPE) - so called due to their composition of distinct protein groups - were recovered and stored in a desiccator containing K₂CO₃ saturated solution (RH = 43.2 %). In the following, the crude protein extract from commercial seeds is referred to as CPE-C and the crude protein extract from pure line seeds as CPE-E.

The procedure for the purification of pigeon pea 7S globulin on a preparative scale was developed by Locali-Pereira et al. (2025) and
performed on a fast protein liquid chromatography (FPLC) system (Akta pure 150, GE Healthcare, Illinois, USA). To produce the 7S globulin purified fraction, only commercial seeds were used, due to their greater availability. Briefly, the crude protein extract was injected into a Q-Sepharose Fast Flow column (GE Healthcare, Illinois, USA) at a flow rate of 13 mL/min. The elution followed two steps: 35 % of 50 mM Tris buffer pH 8.0 + 1 M NaCl solution for a time corresponding to 750 mL of elution volume and 100 % of 50 mM Tris buffer pH 8 + 1 M NaCl solution for a time corresponding to 750 mL of elution volume.

Then, the 7S fractions were pooled and injected directly into a Cellufine GCL 2000 gel filtration column at a flow rate of 12 mL/min, for further purification. The column was equilibrated with ammonium carbonate buffer pH 8 (1 g L⁻¹). After this step, the fractions were pooled, frozen, and freeze-dried for 72 h. The purification yield was approximately 23 % (g of 7S globulin in the purified fraction/g of soluble 7S globulin in the initial flour) (Locali-Pereira et al., 2025).

## 2.3. Composition

The nitrogen content was determined in a Dumas analyser (Thermo Fischer Scientific, Massachusetts, USA). For the crude protein extracts, a nitrogen-to-protein conversion factor of 5.83 was used (Alamanou & Doxastakis, 1997) and for the purified 7S globulin a factor of 5.85 was used, calculated from the amino acid composition of the samples. The lipid content was determined using a extraction method described by Bligh & Dyer (1959) and Folch et al. (1957), using a chloroform/methanol mixture as extraction solvent, a methodology that has proved well-suited to extracting all lipids in pulse protein ingredients (Keuleyan et al., 2023; Koomen et al., 2025). Due to limited material availability, lipid quantification of the purified fraction was carried out on another batch of the same sample, prepared using a dialysis step in place of the desalting step described in Section 2.3; this modification does not affect the sample composition. All samples were measured in triplicate and the protein content was expressed on a dry basis, after determining the moisture content of the samples using the gravimetric method under a nitrogen atmosphere (TGA 2050; TA-Instruments, Delaware, USA).

## 2.4. Fluorescence measurements

Three-dimensional fluorescence measurements (excitation-emission fluorescence matrices, 3D-EEFM) were performed in a spectrofluorimeter (FP-8550, Jasco, Tokyo, Japan) using the front-surface mode. Protein powders were placed in a powder cell and gently compacted. Measurements were performed over an excitation wavelength range from 240 to 480 nm, and an emission wavelength range from 280 to 520 nm. An acquisition step of 1 nm and a bandwidth of 2.5 nm were set for excitation and emission.

## 2.5. Protein dispersions

Samples were dispersed according to protein content (% w w⁻¹) in 20 mM phosphate buffer (pH 8.0) with NaCl adjusted to an ionic strength of 500 mM (Vondel et al., 2023). High ionic strength was employed to ensure adequate solubilization and stability of the protein samples during functional testing. This was necessary to prevent phase separation and to improve reproducibility, particularly for crude extracts. Dispersions were stirred for 2 h at room temperature. Total protein and soluble protein fractions were used in the experiments. For analysis of soluble proteins, samples were centrifuged (10000 g × 10 min x 20 °C) after stirring, with collection of the supernatant.

## 2.6. SDS-PAGE

The protein composition of the crude protein extracts and purified 7S globulins was evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing and reducing conditions with 2-β-mercaptoethanol (5 %, v v⁻¹). About 100 µL of each sample (1 mg total proteins mL⁻¹) were diluted in 100 µL of LDS (2x). The tubes were heated for 5 min at 95 °C. After heating, an aliquot of 15 µL of each sample was added to the wells of the electrophoresis gel. The electrophoresis tank was filled with MES SDS running buffer (Novex) and the experiment was conducted under a voltage of 200 V and 100 mA until complete migration of the samples. After migration, the gel was added to a container with fixation solution (2 % phosphoric acid, 50 % ethanol) and kept under agitation for 30 min. Then, the solution was discarded, and the same procedure was repeated. After the fixation step, the wash solution (17 % ethanol, 15 % ammonium sulfate, 2 % phosphoric acid) was added and the gel was shaken for 30 min. After washing, the sensitization solution was added under agitation for 20 min with the addition of 100 mg of Coomassie Brilliant Blue G 250 dye (Serva) for proteins coloration. Then, the gel was kept under agitation overnight and scanned.

## 2.7. Particle size distribution

The size distribution of protein suspensions was analyzed using dynamic light scattering (DLS) in a Zetasizer Nano ZS (ZEN3600, Malvern Instruments Ltd., Worcestershire, UK). Solutions with 1 % (w w⁻¹) soluble protein were filtered over a 0.2-µm syringe filter. The size distribution was determined by adding 0.7-mL aliquots into cuvettes and equilibrating the samples for 120 s at 20 °C before analysis. The refraction index used was 1.45 for protein dispersions. The size distribution measurements were performed in automatic mode in 10 scans. All measurements were at least performed in triplicate at 20 °C.

## 2.8. Surface tension and surface dilatational properties

The mechanical properties of the air-water interfacial films were studied by performing surface dilatational rheology in a drop tensiometer (TRACKER™ Standard drop tensiometer, Teclis Scientific, Civrieux d'Azergues, France). Soluble protein solutions were evaluated at 0.1 and 1 g L⁻¹. The solutions were loaded in a syringe connected to a straight needle. An 18-Gauge needle was used for all experiments, with 0.84 mm of internal diameter and 100 mm of length. A pendant droplet with a surface area of 10 mm² was formed at the tip of the needle and the surface tension was calculated by fitting the Young-Laplace equation to the shape of the droplet. Before analyses, the droplets were equilibrated for 18000 s, to allow proteins to migrate to and adsorb at the interface. Amplitude sweeps were performed at a constant frequency of 0.02 Hz and an amplitude of 10, 20, and 30 % deformation. In these oscillatory deformations, five cycles were performed for each amplitude step. All measurements were performed at least in triplicate at 20 °C. The raw data were used to calculate the surface pressure (π) and area deformation (ΔA), according to equations (1) and (2) below.

$$ \pi = \gamma - \gamma_0 \qquad (1) $$

$$ \Delta A = \frac{(A - A_0)}{A_0} \qquad (2) $$

where γ and γ₀ are the surface tension of deformed and non-deformed interface, respectively, and A and A₀ are the area of deformed and non-deformed interface, respectively. The data were shown as Lissajous plot by plotting the variation in surface pressure against the applied deformation (Sagis & Fischer, 2014). The elastic (E') and the storage (E') moduli were calculated using the equipment's software. The loss tangent was calculated as the ratio of E'/E'. All plots and calculations were generated using the middle three oscillations.
## 2.9. Foaming properties

Foaming properties were evaluated using the two-syringe method (Fameau et al., 2021; Zhang et al., 2015). Samples were evaluated as total and soluble protein solutions (4 g L⁻¹). Whey protein isolate (WPI) and bovine serum albumin (BSA) were used as controls for good foaming properties and lysozyme (LYZ) was used as control for poor foaming properties. A 2-mL aliquot of each sample was added to 5-mL syringes. The syringe containing the protein solution was connected to another syringe containing 4 mL of air through a tube of 1 mm diameter. The foam was formed inside the syringes by making back and forth movements manually for 15 s. Then, the syringe containing the foam was disconnected, capped, and inverted onto a holding support in front of a black background. Images were captured every 1 min by a camera (EOS, Canon, Tokyo, Japan). The total experiment time was 120 min.

The ImageJ software (NIH Image, Maryland, USA) was used to process raw foam images and determine the foamability, foam stability and volume of drained liquid.

The foam (white) was distinguished from the empty spaces (black) of the syringe by tuning the threshold of the gray values of pixels. The foamability (%) was calculated as the ratio between the volume occupied by the foam immediately after production (t = 0 min) and the total volume of the syringe. The foam stability and the volume of drained liquid were evaluated in a similar way, considering the following times: 1, 3, 5, 10, 15, 30, 45, 60, 90 and 120 min. All measurements were done in triplicate.

## 2.10. Statistical analysis

Data were analyzed using the OriginPro 2016 software. Variance analysis (ANOVA) followed by Tukey post hoc test was applied to determine the difference between samples at a 5 % significance level. The experiments were carried out in three independent replicates.

# 3. Results and discussion

## 3.1. Characterization of crude protein extracts and purified fractions

### 3.1.1. Composition

Both crude protein extracts had a protein content of around 82 wt%, while the 7S globulin fraction had a protein content of around 89 wt% (Table 1). Kornet et al. (2021) obtained a pea protein isolate with a protein content of around 88 wt% (nitrogen-to-protein conversion factor = 5.7) using a mild extraction process combined with diafiltration. Regarding non-proteinaceous compounds, the crude protein extracts showed substantial lipid content, around 8.2–8.5 wt%, whereas only 0.12 wt% were detected in the purified fraction, indicating that the purification process achieved a lipid removal efficiency close to 99 %. The lipid content found in crude protein extracts from pigeon pea is similar to the values reported in study of Keuleyan et al. (2023) for pea and lupin protein concentrates, which had levels around 5 and 9 wt%, respectively. High lipid contents were also reported for pigeon pea protein concentrates using an extensive extraction method

<caption>**Table 1**</caption>

Protein and lipid content of crude protein extracts and purified 7S globulin from pigeon pea seeds.

<table><thead><tr><td>Sample</td><td>Protein content (%, d.b)</td><td>Lipid content (%, d.b)</td></tr></thead><tbody><tr><td>CPE-C</td><td>81.9 ± 0.9<sup>b</sup></td><td>8.2 ± 0.3<sup>a</sup></td></tr><tr><td>CPE-E</td><td>82.2 ± 0.5<sup>b</sup></td><td>8.5 ± 0.2<sup>a</sup></td></tr><tr><td>7S</td><td>89.3 ± 0.2<sup>a</sup></td><td>0.12 ± 0.06<sup>b</sup></td></tr></tbody></table>

Different letters indicate difference between samples (p < 0.05). Crude protein extract obtained from commercial seeds (cv. unknown), crude protein extract obtained from pure line seeds (cv. g18-95), and pigeon pea 7S globulin are indicated by CPE-C, CPE-E, and 7S, respectively.

(Locali-Pereira et al., 2024). Our previous results also showed around 13 wt% of phospholipids in relation to the total lipid content of pigeon pea protein concentrates, which must be taken into consideration when determining interfacial/foaming properties of these samples, since phospholipids may also adsorb at the air-water interface. A high lipid content in protein concentrates has been related to decreases in surface dilatational and shear moduli (Yang & Sagis, 2021). Regarding the other minor compounds in the purified fraction, about 3 % correspond to non-starch polysaccharides, as previously determined (Locali-Pereira et al., 2025). The remainder is expected to correspond to salts (not quantified due to the low sensitivity obtained in TGA analysis).

### 3.1.2. Detection of intrinsic fluorophores in protein extracts

The occurrence, fluorescence patterns and intensity of endogenous fluorophores present in the flours, crude protein extracts, and purified 7S globulins were evaluated by three-dimensional excitation-emission fluorescence matrices (3D-EEFM) in front-surface mode on the dry powders (Fig. 1). The 3D-EEFM showed that both pigeon pea flours presented two different regions of fluorescence: one referring to the protein compounds ($\lambda_{Ex}$ = 270–290 nm, $\lambda_{Em}$ = 300–360 nm corresponding mainly to the tryptophan/tyrosine emission), and another region referring to the fluorescence emissio of non-protein compounds ($\lambda_{Ex}$ ≈ 400–440 nm, $\lambda_{Em}$ = 450–500 nm). While the region of non-protein compounds showed similar fluorescence intensity for both flours, the protein region showed a significant difference, with pure line seeds flour showing a higher fluorescence intensity than commercial seeds flour. Considering that the flours have similar protein content (≈20 %), this result suggests a possible difference in the content, exposure and chemical environment of tryptophanyl residues between the samples. In crude protein extracts, an effective elimination of non-protein endogenous fluorophores ($\lambda_{Ex}$ ≈ 400–440 nm, $\lambda_{Em}$ = 450–500 nm) was noted in both samples compared to their respective flours. Such compounds were removed in the desalting step of the protein extracts. The second region of fluorescence is attributed to the emission region of several phenolic compounds (Locquet et al., 2018). Some phenolic compounds can affect the chemical state of protein that could in turn affect their interfacial properties. However, with the process used in this work and as evidenced by the fluorescence measurements, the significant presence of phenolic compounds in the protein fractions is not expected.

### 3.1.3. Protein composition and size distribution

The protein composition assessed by SDS-PAGE showed different bands, depending on the sample (Fig. 2). SDS-PAGE showed several bands in the CPE-C and CPE-E samples, with four main bands being identified: two bands between 50 and 75 kDa and two bands between 37 and 50 kDa. Two bands with molecular weight between 100 and 150 kDa were also visible in both samples, with the band closer to 100 kDa being more visible in reducing conditions. Several bands were identified with molecular weight <37 kDa, mainly in the presence of a reducing agent. The 7S globulin showed a band between 50 and 75 kDa and two bands close to 50 kDa, characteristic of the α- and β-chains of pigeon pea 7S globulin, respectively. The protein band close to 100 kDa was absent in the 7S globulin without the presence of reducing agent and barely visible when the reducing agent was added. Similarly, bands of molecular weight <37 kDa were also not visualized in non-reducing conditions and visualized at low intensity in reducing conditions. The results show the absence of aggregates around 100 kDa and of low molecular weight compounds (<37 kDa), in the 7S globulin. Although crude extracts consist mainly of 7S globulins, other proteins are present in the samples, such as albumins, protease inhibitors, and 11S globulin (Locali-Pereira et al., 2025).

The hydrodynamic radius of protein molecules affects their diffusion towards the interface, being an important parameter to be considered for studies of interfacial properties (Shrestha et al., 2008). The size distribution of the samples showed a bimodal distribution for the crude
<figure><img src="image_5.png" /><figcaption>**Fig. 1.** Three-dimensional excitation-emission fluorescence matrices of flours, crude protein extracts and purified 7S globulin from pigeon pea seeds. (A) pigeon pea flour from commercial seeds (cv. unknown), (B) crude protein extract from commercial seeds, (C) pigeon pea flour from pure line seeds (cv. g18-95), (D) crude protein extract from pure line seeds, (E) pigeon pea 7S globulin. The color tone corresponds to the fluorescence intensity (AU), see legend on the right-hand side. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** SDS-PAGE of crude protein extracts and purified 7S globulin from pigeon pea with (A) and without (B) reducing agent 2-β-mercaptoethanol. The crude protein extract from commercial seeds (cv. unknown), crude protein extract from pure line seeds (cv. g18-95), 7S globulin are indicated by CPE-C, CPE-E, and 7S, respectively.</figcaption></figure>

protein extracts and the 7S globulin purified fraction with a smaller population around 15 nm and another larger population around 175 nm (Fig. 3). The smaller population was around 17 nm in size for both crude protein extracts. On the other hand, the purified 7S globulin showed

<figure><img src="image_7.png" /><figcaption>**Fig. 3.** Size distribution of crude protein extracts and purified 7S globulin from pigeon pea. The crude protein extract from commercial seeds (cv. unknown), crude protein extract from pure line seeds (cv. g18-95), and 7S globulin are indicated by the green, orange, and blue lines. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>
values around 15 nm. A greater contribution of the smaller population is expected for the purified 7S globulin fraction relative to the crude extracts, as a size of 10–12 nm is characteristic of 7S globulin (Ruiz-Henestrosa et al., 2007). The largest population presented a size of around 175 nm for CPE-C and CPE-E, while the 7S globulin presented value around 153 nm, highlighting a certain degree of aggregation of the proteins in the suspensions. However, there is no significant change in the distribution of the hydrodynamic radius, so that the difference in diffusion to the air-water interface should not be a discriminant factor. Surface charge and hydrophobicity were not determined in this work due to the heterogeneous nature of the systems, although these parameters could play a role in adsorption kinetics, especially in soluble fractions.

## 3.2. Air-water interfacial properties

### 3.2.1. Adsorption behavior

The air-water interfacial properties of crude protein extracts and purified 7S globulin fraction were evaluated at concentrations of 0.1 and 1.0 g L⁻¹. To form stable foams, proteins must diffuse from the bulk solution to the interface and switch to an adsorbed state, orienting their hydrophobic parts towards the air phase and their hydrophilic parts towards the water phase (Graham & Phillips, 1979). Fig. 4 shows the adsorption of proteins at the air-water interface over 3 h. The adsorption of CPE-E was faster among all samples, followed by CPE-C and the 7S globulin. In all experiments, the surface tension value at the first measurement point ranged between 26 and 36 mN/m, indicating that substantial adsorption already took place during the drop formation. The involved mechanisms below 1 s are difficult to describe, given the rapid expansion of the drop and the potential convective, rapid adsorption taking place then. Nevertheless, contrasted behaviors between samples can be observed after the drop target area is reached (i.e., from 1 s on). For instance, at low concentrations (0.1 g L⁻¹) (Fig. 4a), a gradual increase in surface pressure was observed, consistent with the initial diffusion and adsorption of surface-active components. In contrast, at higher concentrations (1.0 g L⁻¹) (Fig. 4b), the surface pressure at the first measurement point was even higher, suggesting that early adsorption events had occurred more rapidly and more completely during the dynamic formation of the drop, bringing the interface closer to saturation within this short sub-second timescale. Nevertheless,

<figure><img src="image_8.png" /><figcaption>Fig. 4. Air-water surface tension over time of pigeon pea protein suspensions at 0.1 g L⁻¹ (A) and 1.0 g L⁻¹ (B). The crude protein extract from commercial seeds (cv. unknown), crude protein extract from pure line seeds (cv. g18-95), and native 7S globulin from pigeon pea are indicated by the colors green, orange, and blue. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)</figcaption></figure>

further adsorption and/or interfacial reorganization still occurred in a later stage, as shown by a decrease in interfacial tension after around 200–300 s. It should be pointed out that even at 0.1 g L⁻¹, the first measurement points already correspond to an interfacial tension substantially lower compared to that of a bare air-water interface (i.e., 72 mN/m). This may be caused by mass transfer events occurring during the rapid formation (and thus dynamic interfacial expansion) of the liquid drop, before the drop volume and surface stabilize, the system then entering quiescent conditions where adsorption becomes driven by diffusion. Lower concentrations (<1.0 g L⁻¹) could be employed to further delay the onset of protein adsorption at the air-water interface during drop formation. From 100 s onwards it is possible to see a clear difference in the reduction in surface tension of both crude protein extracts at low concentration, with CPE-E leading to a faster reduction in surface tension than CPE-C. At high concentration, it takes around 1000 s for the purified 7S globulin to reduce the surface tension to levels similar to those of crude protein extracts. The faster adsorption of crude protein extracts at the air-water interface compared to purified 7S globulin, could be due to the presence of proteins of low molecular weight and/or small non-proteinaceous compounds, which consequently tend to migrate to the interface more quickly. Also, compounds such as saponins or phospholipids may be present in plant protein extracts and it can compete and/or interact with proteins at the interface, modifying their surface properties (Bock et al., 2021; Keuleyan et al., 2024).

In the $π$ versus $t^{0.5}$ ($s^{0.5}$) plots, a linear region was observed in the initial stage of dynamic protein adsorption, implying that this process is primarily driven by diffusion. Due to the linear region for samples on the $π-t^{0.5}$ plot being ended in 20 $s^{0.5}$, the $K_{ads}$ of each sample was calculated through linear regression analysis (before 20 $s^{0.5}$) (Perez et al., 2009; Zhang et al., 2025) (Supplementary Fig. 1) and listed in Table 2. A higher $K_{ads}$ value corresponds to a more rapid adsorption of the surfactant at the air-water interface (Zhang et al., 2025). The results indicate that, at low concentrations, the purified 7S globulin and CPE-C exhibited slightly higher $K_{ads}$ at the air-water interface (around 0.04 mN/m$^{-1}$ s$^{-0.5}$) compared to CPE-E (0.03 mN/m$^{-1}$ s$^{-0.5}$) (Table 2), suggesting that CPE-E may contain components that hinder protein adsorption at the interface, such as aggregates. At higher concentrations, all samples showed increased $K_{ads}$, with the purified 7S globulin reaching the highest value (0.13 mN/m$^{-1}$ s$^{-0.5}$), followed by the crude protein extracts (≈0.11 mN/m$^{-1}$ s$^{-0.5}$). This suggests that, at higher concentrations, the greater purity of the 7S globulin promotes a more efficient and mobile interfacial organization, while the crude protein extracts, possibly due to the presence of non-protein components or proteins with different structures, exhibit slightly restricted interfacial mobility. Thus, the purity and composition of the samples significantly influence the adsorption behavior of proteins at the interface.

### 3.2.2. Surface dilatational rheology

In all conditions evaluated, a decrease in the storage modulus $E'$ values was observed with increasing the deformation amplitude (Fig. 5). This indicates that the microstructure of the interfacial film was

<caption>Table 2</caption>

Adsorption rate ($K_{ads}$, mN m$^{-1}$ s$^{-0.5}$) of pigeon pea protein extracts on air-water interface at different protein concentrations.

<table><thead><tr><th rowspan="2"></th><th colspan="2">Protein concentration (g L<sup>-1</sup>)</th></tr><tr><th>0.1</th><th>1.0</th></tr></thead><tbody><tr><td>7S</td><td>0.041 ± 0.002<sup>a</sup></td><td>0.131 ± 0.006<sup>a</sup></td></tr><tr><td>CPE-C</td><td>0.039 ± 0.005<sup>a</sup></td><td>0.112 ± 0.012<sup>b</sup></td></tr><tr><td>CPE-E</td><td>0.030 ± 0.003<sup>b</sup></td><td>0.114 ± 0.008<sup>b</sup></td></tr></tbody></table>

Different letters in the same column indicate difference between samples ($p < 0.05$). Purified 7S globulin, crude protein extract from commercial seeds, and crude protein extract from pure line seeds (cv. g18-95) are indicated as 7S, CPE-C, and CPE-E.
<figure><img src="image_9.png" /><figcaption>**Fig. 5.** Surface dilatational moduli versus deformation amplitude (A and C), and the loss tangent (B and D) of pigeon pea protein suspensions at 0.1 g L⁻¹ (in the top) and 1.0 g L⁻¹ (in the bottom). The storage modulus (E') and the loss modulus (E'') are represented by full symbols and empty symbols respectively. Symbols indicate different samples: crude protein extract from commercial seeds (cv. unknown) (▲), crude protein extract from pure line seeds (cv. g18-95) (▼), and pigeon pea 7S globulin (●).</figcaption></figure>

impacted by high deformations, being classified as a non-linear visco-
elastic behavior. For protein suspensions at 0.1 g L⁻¹, purified 7S
globulin led to the highest reduction in moduli from 21 to 15 mN/m,
while the films formed from crude protein extracts CPE-C and CPE-E
showed similar reductions of 18 to 13 mN/m (Fig. 5a). The higher
storage modulus of the interface with purified 7S globulin under high
deformation, compared to that with the crude protein extracts, implies
the formation of a more rigid interfacial film, displaying greater resis-
tance to deformation. Consequently, the lower values obtained for the
crude protein extracts suggest that there is less perturbation of the
surface microstructure compared to purified 7S globulin. Similarly, for
protein suspensions at 1 g L⁻¹, the 7S globulin-based films had the
highest reduction in moduli from 20 to 14 mN/m, followed by the two
crude protein extracts, CPE-C and CPE-E, which showed similar values
of decrease from 9 to 7 mN/m (Fig. 5c). Comparatively, pea and
bambara groundnut protein concentrates showed elastic moduli in the
range of 15–30 mN/m (Yang, Berton-Carabin, et al., 2022; Yang & Sagis,
2021; Hinderink et al., 2020). In relation to animal proteins, the elastic
moduli for WPI- and egg white protein-based films reported by Shen
et al. (2024) were considerably higher than systems stabilized by mildly
extracted lentil protein isolate, with values around 110 and 80 mN/m,
respectively. According to the authors, the elastic moduli of animal
proteins showed higher dependence on deformation, compared to
plant-based proteins, with the WPI-stabilized interface being more dis-
rupted by large dilatational deformations than lentil protein-stabilized

interface.

Regarding the loss tangent (E''/E'), all samples presented values <
1.0, which indicates predominantly elastic behavior. In the case of
suspensions at 0.1 g L⁻¹, all samples presented very low loss tangent
values, ranging from 0.08 to 0.27, indicating that all interfaces pre-
sented highly elastic responses to deformations (Sagis & Fischer, 2014)
(Fig. 5b). In the case of suspensions at 1.0 g L⁻¹, the purified 7S globulin
showed a low loss tangent range as well (0.19–0.32), but the crude
protein extracts showed higher loss tangents, with values of 0.56 for
CPE-C and 0.73 for CPE-E, regardless of the applied deformation
(Fig. 5d). The fact that the loss tangent observed for the crude protein
extracts was fairly constant indicates that the elastic contribution is
independent of the applied deformation. The fact that crude protein
extracts have a higher loss tangent value indicates that the interface
formed has a lower tendency to relax to its initial organization when the
deformation stops, which can be, for instance, an indication of a lower
connectivity of the interfacial network.

The amplitude sweeps studies carried out show that at low concen-
trations, purified 7S globulin and crude protein extracts lead to air-water
interfacial films with a similar rheological behavior, probably due to the
predominance of 7S globulins at the interface. However, with increasing
concentration, crude protein extracts begin to behave differently at the
air-water interface compared to the purified fractions, suggesting that
with increasing concentration, other compounds alter the interfacial
composition and structure (other groups of proteins or non-protein
compounds), making it less rigid. The purified 7S globulin led to a more rigid interface, independently of the bulk concentration. Keuleyan et al. (2024) also observed this by evaluating the interfacial properties of pea protein concentrates. The authors investigated the effect of lipids, mainly phospholipids, present in protein concentrates on the interfacial properties of these ingredients. It was observed that at low bulk concentrations, lipids have little contribution to the formation of the interfacial film, while at high bulk concentrations, lipids form distinct domains at the interface, resulting in lower connectivity and mechanical strength of the interfacial film when subjected to deformation. Furthermore, the lipid content reported by the authors (around 12 wt%) for pea protein isolate is slightly higher than that determined in the present work for crude pigeon pea protein concentrates (approximately 8 wt%). This confirms that although these ingredients are referred to as protein-rich, they also have significant levels of lipids. These findings demonstrate the important role of high surface-active minor compounds in the interfacial properties of plant protein ingredients and suggest that the bulk concentration is a key factor in modulating whether these compounds act synergistically or interfere with protein functionality.

### 3.2.3. Lissajous plots

The Lissajous curves represent the response in surface pressure change upon deformation of the formed interfacial film during the expansion (top of the curve) and compression (bottom of the curve) cycles (Fig. 6). A completely viscous, completely elastic and viscoelastic response are indicated by Lissajous plots that have the shape of a circle, a straight line and an ellipse, respectively. The angle formed between the overall slope of the plot and the horizontal axis corresponds to the interfacial stiffness and an axis more inclined to the vertical suggests a stiffer interface (Sagis & Scholten, 2014; Yang et al., 2023). This representation is therefore complementary to the calculation of the moduli and can be helpful to capture interfacial features leading to a non-sinusoidal response upon dilatational deformation, as this then results in asymmetric Lissajous curves.

For suspensions with a concentration of 0.1 g L⁻¹, the Lissajous plot showed similar widths for all samples except CPE-E, indicating a similar dissipation energy after deformation for CPE-C and the 7S globulin. For suspensions with a concentration equal to 1.0 g L⁻¹, the Lissajous plots also showed similar widths for all samples except for CPE-E, in a similar way to what was observed at low concentration. At 30 % deformation,

purified 7S globulin showed slight asymmetry, while samples CPE-C and CPE-E showed more symmetrical behavior even at high deformation.

The purified 7S globulin also led to a plot with a greater inclination towards the vertical axis compared to the plots for crude protein extracts, which presented an inclination more towards the horizontal axis. The presence of more horizontal curves for crude protein extracts when evaluated at high protein concentration indicates that the changes in surface tension is moderate/low. This means that the changes in surface tension induced by the change in area can be rapidly compensated for. The fact that the viscoelastic modulus is lower at 1 g L⁻¹ than at 0.1 g L⁻¹ suggests that the composition of the interfacial film is bulk concentration-dependent. This could be linked to there are small surface-active molecules that can rapidly adsorb to and desorb from the interface during oscillations (Yang & Sagis, 2021). Thus, at higher concentration it is observed a higher relative importance of these molecules compared to large and slower proteins. The greater inclination angle observed for purified 7S globulin at 1 g L⁻¹ suggests the formation of a more elastic and rigid interfacial film. This enhanced rigidity may improve foam stability by resisting bubble coalescence. Fig. 7 presents a schematic overview that summarizes the effect of concentration on the behavior of dispersions of crude protein extract and purified 7S globulin.

## 3.3. Foaming properties

### 3.3.1. Foamability

The foamability of crude protein extracts and purified 7S globulin was evaluated using total and soluble protein suspensions. Using only soluble proteins increased the foamability of the two crude protein extracts (Fig. 8) and LYZ, compared to the total protein suspension. No difference in foamability was observed between the use of total or soluble protein suspensions for purified 7S globulin, BSA and WPI (p > 0.05). This dichotomy may be related to the presence of aggregates in the total protein suspensions of the former samples, which can reduce the adsorption of proteins at the interface and, consequently, their foamability. This becomes even more relevant for the crude protein extracts, as they have not undergone further purification steps, such as aggregate reduction by size-exclusion chromatography. The SDS-PAGE analysis (Fig. 2) supports this, as the crude protein extracts showed protein bands between 100 and 150 kDa that were not observed in the purified 7S globulin. The similar performance of the purified 7S globulin

<figure><img src="image_10.png" /><figcaption>**Fig. 6.** Lissajous plots showing the change in surface pressure as a function of 10, 20 and 30 % deformation of the drop area with pigeon pea protein suspensions at 0.1 g L⁻¹ and 1.0 g L⁻¹.</figcaption></figure>
<figure><img src="image_11.png" /><figcaption>Fig. 7. Schematic overview of the behavior at the air-water interface of dispersions of crude protein extract and purified 7S globulin at low (0.1 g L⁻¹) and high (1.0 g L⁻¹) concentrations.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>Fig. 8. Foamability of crude protein extracts and purified 7S globulin from pigeon pea. Gray bars indicate total protein suspensions (4 g L⁻¹) and white bars indicate soluble protein suspensions (4 g L⁻¹). Uppercase letters indicate differences between total protein suspensions and soluble protein suspensions for a same sample and lower case letters indicate differences between samples for total protein suspensions or soluble protein suspensions (p < 0.05).</figcaption></figure>

with the control proteins BSA and WPI stands out, indicating a foamability similar to the proteins of animal origin evaluated. There was no difference between the foaming capacity of the crude protein extracts (p > 0.05), which presented values slightly below the purified 7S globulin. LYZ used as a negative control, was the sample that showed the lowest foamability.

### 3.3.2. Foam stability

3.3.2.1. Appearance of foams. The physical appearance of the foams was evaluated based on the macroscopic homogeneity or heterogeneity of the bubbles immediately after production and after 120 min of observation (Fig. 9). After production, the physical appearance of the foams produced with soluble protein suspensions was more homogeneous than that produced with total protein suspensions, which presented some larger bubbles that could be observed mainly in the crude protein extracts (Fig. 9a). The BSA and WPI controls showed very small bubble sizes immediately after production in both types of suspension, having the highest homogeneity among the samples. On the other hand, LYZ led to very heterogeneous bubble sizes since its production, in both cases. After 120 min of storage, the ingredients based on pigeon pea proteins led to foams with greater homogeneity in relation to the shape and size of the bubbles for soluble protein suspensions, while the foams produced with total protein suspensions looked more heterogeneous (Fig. 9b). It is also noted that for soluble protein suspensions, the foam prepared with pigeon pea crude extract looked very similar to the WPI-based foam after 120 min of storage.

3.3.2.2. Foam stability and liquid drainage. Different destabilization mechanisms can occur over time, such as liquid drainage, coalescence or Oswald ripening (Vaclavik & Christian, 2008). The stability of the formed foams was evaluated by monitoring the foam layer and the volume of liquid drained over time (Fig. 10). For foams formed with total protein suspensions, the purified 7S globulin led to the most stable system after 120 min of experiment (65 %), followed by WPI (61 %), CPE-E (51 %), BSA (40 %), CPE-C (36 %), and LYZ (18 %) (Fig. 10a). On the other hand, foams formed using soluble protein suspensions showed a stability of 69 % for N-7S, 59 % for WPI, 48 % for CPE-E, 39 % for CPE-C, 30 % for BSA and 20 % for LYZ (Fig. 10b). Akintayo et al. (1999) and Adenekan et al. (2018) observed a foam stability after 120 min of about 50–54 % and 55 % for pigeon pea protein concentrate and isolate,
<figure><img src="image_13.png" /><figcaption>**Fig. 9.** Physical appearance of the foams after production (t = 0 min) and after 120 min of storage for total (A) and soluble (B) protein suspensions. Crude protein extract from commercial seeds (cv. unknown), crude protein extract from pure line seeds (cv. g18-95), pigeon pea 7S globulin, bovine serum albumin, whey protein isolate, and lysozyme are indicated by CPE-C, CPE-E, 7S, BSA, WPI and LYZ, respectively.</figcaption></figure>

respectively.

The type of protein suspension did not significantly impact the observed stability (p > 0.05), except for BSA, which had stability reduced by 10% when using soluble protein suspensions. This difference may be explained by the loss of aggregates that contribute positively to the foam stability formed by BSA (Rullier et al., 2008). The volume of liquid drained over time showed a similar trend for all samples, which at the end of 120 min presented around 60–70 % of their initial volume of liquid drained, both for total and soluble protein suspensions (Fig. 10c–d). Around 50 % of the liquid volume was drained for all samples after 40–45 min, except for LYZ, which since the formation of the foam has already shown liquid drainage of around 60 %. By including LYZ as a negative control, it was demonstrated that the observed foaming behavior was not a general property of all proteins, but rather specific to the structural and compositional characteristics of the samples under investigation. Thus, the results emphasize the functional relevance of the pigeon pea proteins in contrast to a protein with minimal foaming ability.

The best performance observed for the foaming properties of purified 7S globulin as compared to crude protein extracts may be related to different aspects:

(i) **Aggregation state:** although the extraction process for both crude and purified extracts was the same and significant changes in their protein conformation are not expected, their aggregation states may differ. Crude protein extracts exhibit a higher degree of aggregation because they have not undergone purification steps that typically remove aggregates, which contributes to the enhanced functionality observed in purified proteins;

(ii) **Compositional heterogeneity:** At high concentrations, crude extracts contain a larger contribution of other compounds, both proteinaceous (small and aggregated proteins) and non-proteinaceous

(e.g., lipids). With this more complex composition, competition at the air-water interface is expected to occur for crude protein extracts, while for purified proteins the 7S globulin proteins have preference for the air-water interface stabilization;

(iii) **Resistance to deformation:** The more heterogeneous interfacial film formed by the crude protein extracts led to lower resistance to deformation. Over time, this reduced rigidity favors liquid drainage and gas loss in foams made from these extracts. These effects are delayed in purified 7S globulin samples due to the formation of a more resistant interfacial film.

Furthermore, the better foam stability presented by the CPE-E in relation to CPE-C suggests a different composition of stabilizing agents present in the samples. The SDS-PAGE analysis indicates a similar protein composition for the protein extracts of the two seed types (Fig. 2), but the composition of non-protein compounds is not well known and can differ significantly depending on cultivars, soil, climatic conditions, etc. Given the already discussed influence that these minor compounds may have on the air-water interface, their identification, quantification and possible implications in different pigeon pea cultivars should be clarified in future work.

The results obtained in this study indicate that pigeon pea protein suspensions can be used as a total or partial substitute for animal-based foaming agents. This approach is relevant for the development of new aerated products such as mousses, cakes, ice creams, and beverages. Understanding the air-water interfacial behavior of food proteins is equally important for establishing precise guidelines when setting processing parameters for baked and aerated products, supporting improved foam management, shorter processing times, and enhanced production efficiency and product quality (Jin et al., 2025). Further studies should be conducted to assess the impact of different processing parameters, such as pH, ionic strength, and temperature, on the
<figure><img src="image_14.png" /><figcaption>**Fig. 10.** Foam stability over time (A and B) and volume of liquid drained (C and D) of total (in the right) and soluble (in the left) protein suspensions. Symbols indicate different samples: crude protein extract from commercial seeds (cv. unknown) (●), crude protein extract from pure line seeds (cv. g18-95) (▼), pigeon pea 7S globulin (●), bovine serum albumin (◄), whey protein isolate (◆), and lysozyme (▶).</figcaption></figure>

production of these aerated food products.

## 4. Conclusion

This study investigated the air-water interfacial and foaming properties of crude and purified protein extracts from pigeon pea seeds. The interfacial properties revealed that the crude protein extracts adsorb more rapidly than the purified 7S globulin fraction, especially at low bulk concentrations, suggesting that other surface-active compounds present in the extracts rapidly migrate to the interface and decrease surface tension. Purified 7S globulin formed more mechanically resistant interfacial films with a more marked elastic contribution, whereas the crude protein extracts led to films with lower resistance to deformation and higher loss tangent, particularly at higher concentrations. This indicates that both the concentration and composition of the extract significantly influence the structure and mechanical properties of the interfacial film. Regarding foaming properties, the use of soluble proteins—rather than total proteins—increased the foamability of the crude extracts but had no effect on the purified 7S globulin, which performed similarly to soluble dairy proteins such as BSA and WPI. The reduced foamability of the crude extracts is attributed to the presence of aggregates, which hinder protein adsorption at the interface. Purified 7S globulin also exhibited the highest foam stability, which may be linked to the ability to forming interfacial films resistant to deformation and liquid drainage. In contrast, the foams prepared with crude extracts showed lower stability due to the greater compositional heterogeneity of the ingredient, including the presence of various components competing at the interface. These findings highlight the potential of pigeon pea proteins as partial or complete substitutes for animal-based foaming

agents in aerated food products.

## CRediT authorship contribution statement

**Adilson Roberto Locali-Pereira:** Writing – original draft, Methodology, Investigation, Conceptualization.
**Joëlle Davy:** Methodology.
**Eléna Keuleyan:** Methodology.
**Véronique Solé-Jamault:** Writing – review & editing, Methodology.
**Valérie Beaumal:** Methodology.
**Renata Tieko Nassu:** Resources.
**Rodolfo Godoy:** Resources.
**Vânia Regina Nicoletti:** Writing – review & editing, Supervision, Conceptualization.
**Claire Berton-Carabin:** Writing – review & editing, Supervision, Conceptualization.
**Adeline Boire:** Writing – review & editing, Supervision, Conceptualization.

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Acknowledgments

The authors thank the Brazilian Federal Agency for Support and Evaluation of Graduate Education (CAPES), the Brazilian National Council for Scientific and Technological Development (CNPq, Proc. 310788/2020-0) and the Ministry of Europe and Foreign Affairs of France (EIFFEL Excellence Program) for the financial support of ARLP's PhD grant. The authors are also grateful to Embrapa for the donation of pigeon pea seeds (cv. g18-95). The authors are grateful to Perrine
Gelebart (INRAE BIA) for kindly performing the analyses to determine
the lipid content in the purified fraction.

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodhyd.2025.112131.

Data availability

Data will be made available on request.

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