Ultrasound‐Extracted Red Grape Seed Protein as a Partial Egg Replacer in Cake Formulation

ABSTRACT

Egg yolk is a key multifunctional ingredient in cakes, contributing to structure, emulsification, foaming, and color, but its replacement with plant‐based proteins is increasingly explored due to sustainability, health, and dietary considerations. This study aims to develop an alternative protein concentrate from red grape seed meal and evaluate its potential as a partial egg replacer in cake production. Red grape seed protein concentrates (GSPs) were produced using conventional alkaline extraction (AE) (pH 10, 15 min) and ultrasound‐assisted alkaline extraction (UAE) (80% amplitude, pH 10, 15 min) and subsequently characterized for protein recovery, water‐ and oil‐binding capacities (WBC and OBC), molecular weight distribution, and structural properties. UAE yielded a higher protein recovery (8.2%) compared to AE (6.7%), while also improving WBC (3.36 ± 0.05 g/g) and OBC (6.49 ± 0.01 g/g). The protein content of both concentrates exceeded 50% (GSP‐AE: 53.48%; GSP‐UAE: 54.37%). SDS–PAGE revealed no major changes in molecular weight distribution but showed increased band intensity by UAE, suggesting enhanced protein recovery and extractability. FTIR analysis indicated ultrasound‐induced conformational modifications, while SEM images revealed smaller and more porous protein particles, which collectively contributed to the improved functional properties of GSP‐UAE. Therefore, the UAE‐derived protein concentrate (GSP‐UAE) was incorporated into cake formulations at 10% and 20% egg yolk replacement levels. Cake quality was evaluated by physical, textural, color, and antioxidant properties. Cakes with 10% substitution showed similar height and hardness to the control, indicating comparable structural quality. At 20% substitution, cakes became harder and more compact, with more pronounced color changes. Antioxidant activity increased dose‐dependently up to 375.25 µg Trolox/g. Overall, the results demonstrate that UAE improves the functionality of GSP and that lower incorporation level enables partial egg yolk replacement without compromising cake quality, while simultaneously enhancing the nutritional and functional value of the product.

Practical Applications

This study shows that protein extracted from red grape seed pomace using ultrasound can be used as a functional, plant‐based ingredient to partially replace egg yolk in cake formulations. At moderate substitution levels, cake quality is maintained while antioxidant content is increased. This approach offers bakeries and food manufacturers a practical way to reduce egg usage and valorize grape processing by‐products in more sustainable bakery products.

1. Introduction

Egg is a key ingredient in cake production, valued both for its nutritional value and for its unique techno‐functional properties in terms of foaming, emulsifying, controlling sugar crystallization, and improving color and flavor attributes. The egg yolk contains approximately 52% dry matter, 31.8%–35.5% lipid, and 15.7%–16.6% protein (Li‐Chan et al. ¹⁹⁹⁵). The multifunctionality of egg yolk in cake formulations is attributed to the distinct structural and physicochemical properties of its constituent protein fractions (Deleu, Melis, et al. ²⁰¹⁷). Egg yolk dry matter is distributed over four major protein fractions: low‐density lipoprotein (LDL, ∼70%), high‐density lipoprotein (HDL, ∼16%), livetins (∼10%), and phosvitin (∼4%) (Li‐Chan et al. ¹⁹⁹⁵). Egg yolk components act as natural emulsifiers due to the lecithin content that stabilize fat–water mixtures in cake batters (Palav, 2015). Protein networks formed during baking provide the structural framework of the cake (Deleu, Melis, et al. ²⁰¹⁷). Furthermore, egg yolk carotenoids—mainly carotene and xanthophylls—contribute to the characteristic yellowish color of the final product (Wu, 2014). Due to this multifunctionality, egg has a critical role in achieving the desirable quality attributes in terms of volume, color, texture, and appearance in cakes.

However, there is a growing need toward identifying effective egg replacements in food systems, motivated by health concerns, allergenicity, evolving dietary preferences toward plant‐based alternatives, and economic considerations (Viana et al. 2023; Yazici and Ozer ²⁰²¹). A variety of ingredients have been investigated as potential egg replacers in cake formulations, including plant‐based (legumes, pseudo‐cereals, fruit/vegetable wastes, and oilseeds) and animal‐based (milk proteins and marine proteins) alternatives as well as hydrocolloids due to their potential effects on batter properties (viscosity, textural, and rheological properties) and cake quality parameters (moisture content, baking loss, specific volume, external properties, color, texture, crumb structure, and sensory properties) (Agregán et al. 2025; Esendemir et al. ²⁰²⁵; Hedayati et al. ²⁰²²; Yazici and Ozer ²⁰²¹). The current egg replacer market has been growing steadily, reflecting an intensified interest in plant‐based baking solutions (Halm et al. 2024). Legumes, pseudo‐cereals, nuts, and fruit/vegetable wastes are among the mostly studied plant‐based protein sources (Burbano et al. 2024; Hariharan et al. ²⁰²³; Hedayati et al. ²⁰²²; Mattila et al. ²⁰¹⁸; Viana et al. ²⁰²³; Yildiz, Karabulut, Karaca, et al. ²⁰²⁵). In particular, underutilized food by‐products such as oilseed and fruit seed meals (the press‐cake residuals after cold‐pressed oil extraction) is a promising class of sustainable protein sources (Burbano et al. 2024; Hariharan et al. ²⁰²³; Singh et al. ²⁰²²; Yildiz, Karabulut, Sıçramaz, ²⁰²⁵).

Grape seeds are one of the by‐products of the food industry. Following cold‐pressing or solvent extraction of grape seed oil, the resulting defatted grape seed meal is often underutilized, typically ending up as waste or low‐value animal feed (Ubaid and Saini 2024). Therefore, it can be valorized within the circular economy principles to produce value‐added protein ingredient that can have the potential to substitute egg yolk in certain food systems. In addition, grape seed meal is rich in bioactive compounds such as proanthocyanidins, anthocyanins, flavonols, and phenolic acids, which provide a certain antioxidant activity (Ghafoor et al. 2020; Sochorova et al. ²⁰²⁰). This composition distinguishes grape seed protein concentrate (GSP) from most other plant‐based protein sources, such as soy, pea, or chickpea, which are primarily valued for their amino acid profile and functional properties with minimal antioxidant contribution. Moreover, grape seed products in the form of grape seed powder or extracts have been increasingly explored as value‐added ingredients to various food formulations such as bread and biscuit (Maman and Yu 2019; Oprea et al. ²⁰²²). Cake formulations were selected in the present study because egg yolk plays multiple technological roles in cake systems, including emulsification, structure formation, moisture retention, texture development, and color formation. Replacing egg yolk in cakes is therefore particularly challenging, making cake systems a suitable model for evaluating the techno‐functional performance of alternative plant proteins. Accordingly, the dual functionality provides a unique advantage for the development of bakery products by serving both as a partial egg substitute and as a natural source of antioxidants.

The functional performance of plant proteins can vary widely depending on their botanical source, extraction method, and formulation environment, and their functionality often remains lower than that of animal‐derived proteins. To achieve desirable product quality, plant proteins may require enhancement through physical, chemical, or enzymatic modification strategies (Akharume et al. 2021; Dhiman et al. ²⁰²³). Ultrasound (US) is a promising process for this purpose. US treatment of protein dispersions induces cavitation that disrupts intermolecular interactions, partially unfolds protein molecules, and modifies particle size, collectively exposing reactive groups and increasing chain flexibility to improve key functional properties such as solubility, emulsification, foaming, and water/oil binding capacity (OBC) (Gharibzahedi and Smith 2020; López‐Mártir et al. ²⁰²⁵).

The objective of the present study is to develop an alternative plant‐based protein concentrate from red grape seed meal and evaluate its potential as a partial egg replacer in cake production. For this purpose, GSP was produced by ultrasound assisted alkaline extraction (UAE) to enhance its techno‐functional properties. It is hypothesized that US‐modified GSP can be used in cake formulation for partial egg replacement without compromising cake quality while simultaneously enriching the product with antioxidant compounds. In this respect, cakes formulated with US‐treated GSP were compared to full‐egg control cake in terms of physical, chemical, and textural properties. Thus, this study addresses both the valorization of an industrial by‐product into a functional food ingredient and the development of egg‐reduced bakery products to meet health, dietary, and sustainability demands.

2. Materials and Methods

2.1. Materials

Red grape seed meal left after cold‐pressed oil production was kindly provided by a local company (TALYA, Sakarya, Turkey) in dry and grounded form. The meal was defatted using 99.5% acetone (Arcan and Yemenicioğlu 2007) to remove potential residual oil and then air‐dried at 25°C and used as defatted grape seed powder (dGS, containing 7.64% protein) in further steps. All chemicals and reagents were purchased from Sigma (Steinheim, Germany) at analytical or higher grade.

2.2. Protein Extraction Procedures

Red GSPs were produced using two methods: ultrasound‐assisted alkaline extraction (UAE) and conventional alkaline extraction (AE) (Yildiz, Karabulut, Sıçramaz, 2025). For UAE, the dGS powder was mixed with distilled water at a 1:10 ratio (w:v) and rehydrated for 1 h at 4°C. The pH of the mixture was then adjusted to 10.0 using 2 N NaOH, and the suspension was subjected to US treatment at 80% amplitude for 15 min of extraction using an ultrasonic probe system (Sonics VCX750, Newtown, USA) equipped with a 13 mm titanium probe. For AE, the same protocol was applied, with the US step omitted. Following extraction, both suspensions were centrifuged at 10000 rpm for 15 min at 4°C to separate the liquid containing the dissolved proteins. The supernatants were then acidified to pH 4.50 to precipitate the proteins, followed by another centrifugation step at 15000 rpm for 10 min at 4°C. The resulting protein pellets were neutralized with 0.1 N NaOH and freeze‐dried (Labconco FreeZone, −45°C, 0.045 bar) to obtain the GSP‐UAE and GSP‐AE powders.

2.3. Determination of Soluble and Total Protein Contents

The soluble protein content in the supernatants obtained after the first centrifugation step (for both UAE and AE) was determined using the Bradford method (Bradford 1976). Briefly, 150 µL of each supernatant was mixed with 3 mL of Bradford reagent (Coomassie Brilliant Blue G‐250) and incubated for 10 min at room temperature. The absorbance was then measured at 595 nm using a spectrophotometer (Shimadzu, UV‐1280, Japan). A standard curve prepared with bovine serum albumin (BSA) was used to calculate the total soluble protein concentration, and results were reported as mean ± standard deviation.

Protein content of GSPs was analyzed by Kjeldahl method with the nitrogen conversion factor of 5.30 (FAO/INFOODS, ²⁰¹²). Protein recovery rate in the GSPs was calculated based on the following equation:

$$\mathrm{Protein}\mathrm{recovery}\mathrm{rate}\left(\%\right)=\frac{m_{\mathrm{GSP}}\times P_{\mathrm{GSP}}}{m_{\mathrm{dGS}}\times P_{\mathrm{dGS}}}\times 100$$

where m _GSP and P _GSP are the weight and protein content of red grape seed protein concentrates, and m _dGS and P _dGS are the weight and protein content of the dGS, respectively.

2.4. Water Binding‐ and Oil Binding‐ Capacity

The water binding capacity (WBC) and oil binding capacity (OBC) of GSP‐AE and GSP‐UAE were measured according to the method of Aydemir and Yemenicioĝlu (2013). Each protein concentrate (50 mg) was mixed with 1.5 mL of either distilled water or sunflower oil and incubated for 30 min at ambient temperature to allow water or oil absorption. The mixtures were then centrifuged at 13500 rpm and 25°C for 20 min, after which the supernatants were discarded and the pellets were weighed. WBC and OBC were calculated as grams of water or oil absorbed per gram of protein.

2.5. SDS‐PAGE Analysis

The molecular weight distribution of GSP‐AE and GSP‐UAE samples was determined by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) according to the method of Brunelle and Green (2014). Electrophoresis was carried out using a Tris–glycine system consisting of a 12% resolving gel and a 5% stacking gel. Protein samples were prepared at 6.7 mg/mL under both reducing and non‐reducing conditions using SDS sample buffer with or without β‐mercaptoethanol, respectively. For non‐reducing conditions, samples were mixed with sample buffer (120 mM Tris‐HCl pH 6.8, 4% SDS, 20% glycerol, 0.05% bromophenol blue) without β‐mercaptoethanol. For reducing conditions, β‐mercaptoethanol was added to the sample buffer at a final concentration of 750 mM to cleave disulfide bonds. All samples were heated at 100°C for 5 min and briefly centrifuged prior to loading. Molecular weight marker and protein samples were loaded into the wells, and electrophoresis was performed at a constant voltage of 200 V until the bromophenol blue dye front reached the bottom of the resolving gel. Following electrophoresis, gels were stained with Coomassie Brilliant Blue R‐250 (0.1% in 50% methanol/6.8% acetic acid) and destained with a methanol (40%)/acetic acid (10%) solution to visualize protein bands. The Bio‐Rad Precision Plus Protein Standards (10–250 kDa) served as the molecular weight marker for band identification.

2.6. Fourier‐Transform Infrared Spectroscopy (FTIR)

FTIR analysis was performed using an infrared spectrometer (PerkinElmer Spectrum Two) with a resolution of 4 cm⁻¹. An air spectrum was recorded as the background and subtracted from all sample spectra. GSP‐AE and GSP‐UAE samples were placed on the measurement plate, and gentle pressure was applied to ensure proper contact. Spectra were collected over the wavenumber range of 400–4000 cm⁻¹, with four scans averaged for each measurement. Both the original FTIR spectra and their second derivatives were examined to assess potential changes in the proteins’ secondary structure.

2.7. Field Emission Scanning Electron Microscopy (FE‐SEM)

Microstructures of protein concentrates were determined using an FE‐SEM (FEI Quanta FEG 450). GSP‐AE and GSP‐UAE samples were coated with gold/palladium layer and affixed to carbon fiber tape. The morphological differences were observed at different magnification sizes (500×, 1000×, and 2000×).

2.8. Cake Preparation

Cakes were prepared following the procedure described by Grossi Bovi Karatay et al. (2022), with some modifications. Three formulations were developed: a control cake containing the full amount of egg yolk (C) and two experimental cakes in which 10% and 20% of the egg yolk were replaced with US‐modified GSP (US10 and US20), as shown in Table 1. For each formulation, sunflower oil and sugar were first blended for 2 min. The emulsifying component (egg yolk for the control sample or US‐modified GSP for the substitution treatments) was then added and mixed for 3 min. Milk and wheat flour were subsequently incorporated and mixed for an additional 3 min to obtain a homogeneous batter. Finally, baking powder was added and blended for 30 s. The resulting batters were portioned into paper molds placed inside a muffin pan and baked in a conventional oven at 180°C for 15 min. After baking, the cakes were removed from the molds and cooled at ambient temperature for approximately 2 h before conducting further analyses.

TABLE 1.

Cake formulations.

	Cake samples
Ingredients	C (%)	US10 (%)	US20 (%)
Wheat flour	31.5	31.5	31.5
Refined sugar	22.8	22.8	22.8
Whole milk	16.1	16.1	16.1
Vegetable oil (sunflower oil)	13.9	13.9	13.9
Baking powder	1.8	1.8	1.8
Egg yolk	13.9	12.5	11.1
Grape seed protein concentrate (GSP)	0	1.4	2.8
Total	100	100	100

Note: C represents the control cake formulated with 100% egg yolk, whereas US10 and US20 refer to the cakes in which ultrasound‐modified GSP replaced 10% and 20% of the egg yolk, respectively.

2.9. Cake Analysis

2.9.1. Cake Appearance, pH, and Moisture Content

To assess the influence of the emulsifier on cake rise, the height of each cake was measured using a digital caliper after cooling the samples. To measure the pH, the cakes were ground using a coffee grinder, and the resulting powder was mixed with distilled water at a 1:9 ratio (w:v). The mixture was homogenized with a mechanical homogenizator (Daihan, HG‐15D, Korea) at 3000 rpm for 1 min, after which the pH was recorded using a calibrated pH meter (Ohaus, AB33M1‐F). The moisture content of the cake samples was determined following the method of Grossi Bovi Karatay et al. (2022). Ground cake samples were placed in a moisture analyzer and dried at 105°C for approximately 15 min. Moisture content was expressed as percentage on a wet basis.

2.9.2. Baking Loss

Baking loss (%) was determined following the method of Grossi Bovi Karatay et al. (2022). For this measurement, the weight of the cake batter was recorded first, and the baked cakes were weighed again after cooling for about 2 h at room temperature. The loss that occurred during baking was calculated from the difference between the initial batter weight and the final cake weight, using the following equation below:

$$\mathrm{Baking}\mathrm{loss}\left(\%\right)=\frac{W_{\mathrm{i}}-W_{\mathrm{f}}}{W_{\mathrm{i}}}\times 100$$

where W _i represents the initial weight of the batter and W _f is the weight of the baked cake after cooling.

2.9.3. Antioxidant Activity

Antioxidant compounds were first extracted from the cake samples using a solvent extraction method adapted from Kowalski et al. (2023) with modifications. Briefly, the cakes were ground into a fine powder and mixed with 70% methanol at a ratio of 1:10 (solid:liquid, w/v). To enhance the extraction efficiency, the mixture was treated in an ultrasonic water bath for 10 min and then stirred on a magnetic stirrer for an additional 15 min. The extracts were subsequently centrifuged at 8000 rpm for 15 min, and the clear supernatant was collected and stored at −20°C until further analysis.

Antioxidant activity was then determined using the DPPH radical scavenging assay described by Brand‐Williams et al. (1995). For this analysis, 150 µL of the cake extract was mixed with 2850 µL of freshly prepared methanolic DPPH solution (0.051 mM). The reaction mixtures were kept in the dark for 30 min to allow the scavenging reaction to occur, after which absorbance was measured at 515 nm using a UV–Vis spectrophotometer (Shimadzu UV‐1280, Japan). The DPPH assay was performed in triplicate. The percentage inhibition values were converted to Trolox equivalents using a calibration curve prepared with Trolox solutions ranging from 10 to 100 µM. Antioxidant activity was finally expressed as µg Trolox equivalent per mg of cake.

2.9.4. Textural Properties

Texture profile analysis (TPA) of the cakes was determined according to the method adapted from Salehi and Kashaninejad (2018) with modifications. Measurements were carried out using a texture analyzer (TexturePro CT3, V1.9 Build 35, Brookfield Engineering Labs Inc., USA) equipped with a 30 mm cylindrical probe. Cake samples were subjected to a double‐compression test, in which each sample was compressed to 50% of its original height at a test speed of 2.0 mm/s. The pre‐test and post‐test speeds were both set at 2.0 mm/s, and a trigger force of 5 g was applied to ensure consistent contact with the sample. The texture characteristics of the cakes were evaluated based on standard TPA parameters, including hardness (g), cohesiveness, springiness, gumminess (g), chewiness (g), and resilience.

2.9.5. Color Properties

The color parameters of the cake samples (L*, a*, and b*) were measured using a colorimeter (Konica Minolta CR‐400, Japan) according to the CIELAB color system. Measurements were taken on both the external and internal surfaces of each cake, with five readings per sample. Total color difference (ΔE) was calculated relative to the control cake using the following equation.

$$\mathrm{\Delta }E=\sqrt{{(\mathrm{\Delta }{L}^{\ast })}^2+{(\mathrm{\Delta }{a}^{\ast })}^2+{(\mathrm{\Delta }{b}^{\ast })}^2}$$

2.10. Statistical Analysis

All analyses were conducted with three replicates. The experimental data were recorded as the mean ± standard deviation and then subjected to an independent‐samples t‐test for protein concentrates and one‐way ANOVA for cake samples at a 95% confidence level using Minitab 16 data analysis software (Minitab Inc.).

3. Results and Discussion

3.1. Characteristics of Grape Seed Protein Concentrates

3.1.1. Protein Content and Recovery

The total soluble protein contents of the liquid extracts collected immediately after the centrifugation step of the extraction processes were determined as 936.27 ± 86.78 and 232.84 ± 4.90 mg/100 g for UAE and AE, respectively (p < 0.05), according to the Bradford protein assay. These findings indicate that UAE markedly enhanced the transfer of proteins from the grape seed meal matrix into the AE medium. The substantially higher soluble protein content observed in UAE samples can be attributed to ultrasonic cavitation, which generates localized shear forces, turbulence, and microstreaming effects that improve matrix disruption and mass transfer, facilitating the release of proteins to the extraction medium (Görgüç et al. 2019; Kadam et al. ²⁰¹⁵; Rahman and Lamsal ²⁰²¹). In addition, US treatment likely promotes partial unfolding and disaggregation of protein structures, exposing previously buried hydrophilic groups and thereby increasing protein–water interactions and solubility (Eze et al. 2022; Wang et al. ²⁰²⁰). US reduces protein particle size and aggregation, leading to better dispersion and solubility in aqueous solutions (Wang et al. 2020; Zhang et al. ²⁰²⁵). Similar improvements in protein solubilization following UAE have been reported for several plant protein systems, including pea pod and pistachio meal proteins (Karabulut et al. 2023; Yildiz, Karabulut, Sıçramaz, ²⁰²⁵).

The protein contents and recoveries are summarized in Table 2 for GSPs. Both extraction methods produced protein concentrates with protein contents of 53.48 ± 2.70% (GSP‐AE) and 54.37 ± 3.08% (GSP‐UAE), with no statistically significant difference (p > 0.05). These values fall well within the protein concentrate range (35‐80%) and below the 90% protein threshold that defines protein isolates (Meganaharshini et al. 2023). These results demonstrate that grape seed meal, despite being an agro‐industrial by‐product with relatively lower protein content, can be effectively enriched to contain > 50% protein, highlighting its potential as a valuable plant‐based protein source. The relatively modest protein content may be attributed to the compact lignocellulosic structure of grape seed press cake, which limits protein extractability, as well as the co‐extraction of non‐protein components. Additional purification processes, including ultrafiltration, microfiltration, or repeated washing steps, may be necessary to obtain protein isolate–level purity. Nevertheless, both GSP‐AE and GSP‐UAE contained protein levels exceeding 50%, representing a substantial enrichment compared to the raw dGS, which contained 7.64% protein.

TABLE 2.

Chemical and functional properties of red grape seed protein concentrates.

Sample	Protein (%)	Protein recovery (%)	WBC (g/g)	OBC (g/g)
GSP‐UAE	54.37 ± 3.08a	8.2 ± 0.46	3.36 ± 0.05a	6.49 ± 0.01a
GSP‐AE	53.48 ± 2.70a	6.7 ± 0.34	2.08 ± 0.03b	5.73 ± 0.02b

Note: GSP‐UAE and GSP‐AE refer to the red grape seed protein concentrates obtained by ultrasound‐assisted alkaline extraction and conventional alkaline extraction, respectively. WBC and OBC indicate the water binding capacity and oil binding capacity, respectively. Different superscript letters within the same column indicate statistically significant differences between the protein concentrates (p < 0.05).

Although the overall protein contents of the concentrates were comparable, the efficiency of protein recovery differed between the extraction methods. The UAE process resulted in a higher protein recovery (8.2%) compared to conventional AE (6.7%), indicating that US treatment better facilitated the release of proteins from the grape seed matrix. This finding was consistent with the higher soluble protein content observed in the liquid extracts after centrifugation as discussed above. US‐generated cavitation helps break down the compact grape seed structure through localized shear forces and turbulence, allowing the alkaline solution to penetrate the matrix more effectively and expose more proteins for extraction. Badjona et al. (2024) reported protein extraction yields of 19.75% using US‐assisted extraction and 16.41% using a conventional method, producing faba bean protein isolates with higher protein purities of approximately 90% due to the applied US conditions and the use of a protein‐rich raw material with relatively lower fiber content. Protein extraction efficiency is known to be strongly influenced by the nature of the protein source, the structure of the plant matrix, and processing conditions such as pH, ultrasonic power, temperature, and extraction time (Karabulut et al. 2023; Wang et al. ²⁰²⁰; Yildiz, Karabulut, Sıçramaz, ²⁰²⁵; Zhang et al. ²⁰²⁵). In materials rich in lignocellulosic components, proteins are often embedded within a rigid structural network, which limits their extractability. Agricultural by‐products such as pea pods, which contain only 10–13% protein and are rich in lignocellulosic materials, present greater challenges for protein recovery. In such matrices, even US‐pretreated AE resulted in protein yields of around 21%, reflecting the limiting effect of structural components (Karabulut et al. 2023). In the present study, the relatively lower protein recovery values can therefore be attributed to several factors, including the moderate initial protein content of grape seed meal, the complex and compact nature of the press cake matrix, and the relatively short sonication times applied. Despite these limitations, UAE clearly enhanced both protein recovery and total soluble protein content compared to AE.

3.1.2. Molecular Weight Distribution

The molecular weight distributions of GSP‐AE and GSP‐UAE under non‐reducing and reducing SDS–PAGE conditions are presented in Figure 1. Under non‐reducing conditions, both samples exhibited a similar pattern of broad, heavily stained bands extending from the top of the resolving gel to approximately 50 kDa. Such smeared profiles are characteristic of heterogeneous protein aggregates stabilized by disulfide linkages and non‐covalent interactions, which limit complete dissociation. Similar aggregation behavior has been reported for plant proteins extracted from polyphenol‐rich matrices, where protein–protein and protein–phenolic interactions can restrict complete dissociation during electrophoresis (Alu'datt et al. ²⁰¹⁹). Under reducing conditions, clearer and more distinct protein bands appeared at approximately 35–40 kDa, 18–22 kDa, and 10–15 kDa, indicating the partial dissociation of aggregated proteins into lower‐molecular‐weight subunits after cleavage of disulfide bonds by β‐mercaptoethanol. Compared with GSP‐AE, the GSP‐UAE samples exhibited greater band intensity, suggesting enhanced protein solubilization and extraction efficiency following US treatment. Importantly, no additional bands appeared and no existing bands disappeared after US treatment, indicating that US did not induce peptide bond fragmentation or severe protein degradation. Instead, US likely promoted the dissociation of high‐molecular‐weight protein aggregates into more soluble subunits. Similar observations have been reported for US‐treated walnut proteins (Zhu et al. 2018) and pistachio meal proteins extracted by UAE (Yildiz, Karabulut, Sıçramaz, 2025), where sonication increased band intensity without substantially altering the molecular weight distribution. Thus, it is inferred that US conditions applied were sufficiently mild to avoid extensive protein degradation while still improving protein extractability and functionality in food systems. Accordingly, in the present study, the stronger bands observed for GSP‐UAE support the higher soluble protein content and protein recovery obtained by UAE.

FIGURE 1.

SDS‐PAGE image for the molecular weight distribution of grape seed protein concentrates (GSPs) obtained by conventional alkaline (AE) and ultrasound‐assisted alkaline extraction (UAE).

3.1.3. Water‐ and Oil‐Binding Capacity of GSPs

The practical value of GSPs in food applications largely depends on their functional behavior, particularly their interactions with water and oil. Properties such as WBC and OBC are strongly influenced by protein solubility, molecular flexibility, and surface characteristics, all of which can be altered by processing conditions. As demonstrated in Table 2, both WBC and OBC were improved by UAE (p < 0.05). WBC and OBC represent the ability of a protein concentrate to absorb and retain water and oil, respectively, per gram of protein. In this regard, GSP‐UAE exhibited a WBC of 3.36 ± 0.05 g water/g protein, whereas GSP‐AE showed a lower WBC of 2.08 ± 0.03 g water/g protein; similarly, the OBC of GSP‐UAE (6.49 ± 0.01 g oil/g protein) was significantly higher than that of GSP‐AE (5.73 ± 0.02 g oil/g protein) (p < 0.05). These improvements can be attributed to US‐induced structural modifications, including the structural modifications and the partial unfolding of polypeptide chains, which expose previously buried hydrophilic groups and hydrophobic residues, thereby increasing the accessible surface area for interactions with water and oil molecules, respectively (Ding et al. 2021; Gul et al. ²⁰²³). Regarding the structural changes, FTIR spectra of GSP‐AE and GSP‐UAE, including original spectra and second‐derivative plots, are presented in Figure 2, demonstrating slight changes in the Amide I (1700–1600 cm^{− 1}, reflecting C = O stretching of the peptide backbone), Amide II (1600–1500 cm^{− 1}, N–H bending and C–N stretching), and Amide III (1350–1200 cm^{− 1}, coupled C–N stretching and N–H in‐plane bending) regions especially with the second‐derivative plot for the FTIR spectra. This is consistent with earlier studies, demonstrating that ultrasonic treatment modifies the functional properties of plant proteins, likely due to the alterations in the hydrophilic groups secondary and tertiary structures (Tang et al. 2021).

FIGURE 2.

The original (a) and second‐derivative (b) FTIR spectra of grape seed protein concentrates extracted by alkaline extraction (GSP‐AE) and ultrasound‐assisted alkaline extraction (GSP‐UAE).

It is worth noting that an increase in WBC upon using novel extraction methods has been seen in other proteins as well. For instance, in a study on moringa oleifera seeds, UAE (75% amplitude, 1:20 solute‐to‐solvent ratio, 11 pH, 20 min) resulted in greater WBC than the conventional extraction (Fatima et al. 2023). Ultrasonic cavitation can lead to smaller protein particles with increased surface area, which can increase the interaction of hydrophilic groups of proteins and water molecules as well as the amount of water that proteins can hold (Ding et al. 2021; Gul et al. ²⁰²³). The SEM micrographs (Figure 3) further support this interpretation, revealing that GSP‐UAE particles exhibited a more porous and fragmented morphology than the larger and more compact aggregates observed in GSP‐AE, thereby increasing the specific surface area available for water and oil interactions. Greater OBC values indicate that the protein has more surfaces or sites that can associate with lipid molecules. The fact that UAE improved OBC suggests that the sonication process may have exposed more hydrophobic (oil‐attracting) regions of the protein or produced smaller protein particles with more surface area for binding lipids. Accordingly, de Medeiros et al. (2025) also demonstrated an improved OBC due to the increased surface hydrophobicity and free sulfhydryl groups.

FIGURE 3.

SEM images of selected grape seed protein concentrates produced by alkaline extraction (GSP‐AE) and ultrasound‐assisted ultrasound extraction (GSP‐UAE).

For both WBC and OBC, our findings were in line with the study of Badjona et al. (2024), where better water and oil binding capacities were achieved after UAE compared to control protein isolate. The enhanced water‐ and oil‐binding capacities suggest that GSP‐UAE is more suitable for applications requiring efficient moisture retention and lipid stabilization. These properties are particularly relevant for its use as a partial egg replacer in cake formulations, where improved water‐binding capacity may support crumb moisture retention during baking, while enhanced oil‐binding capacity may facilitate fat stabilization in the batter and contribute to the formation of the final cake structure. Although grape seed proteins differ from egg yolk proteins in composition, SDS–PAGE revealed the presence of protein fractions predominantly distributed between 10 and 40 kDa, a molecular weight range commonly associated with functional plant proteins capable of participating in water binding, oil binding, and heat‐induced network formation. Together with the enhanced WBC and OBC values, these characteristics may contribute to the ability of GSP‐UAE to partially replace certain technological functions of egg yolk in cake formulations.

3.2. Cake Properties

3.2.1. Cake Appearance, pH, and Moisture Content

The visual appearance of the cake samples is presented in Figure 4. The final cake height was comparable between the control cake and the cake containing 10% US‐modified grape seed protein (US10), indicating that partial replacement of egg at this level did not noticeably affect cake expansion during baking. In contrast, increasing the level GSP to 20% (US20) resulted in a denser crumb structure and a reduction in cake height (p < 0.05) (Table 3). Such changes are commonly observed when animal‐based ingredients like eggs are partially replaced with plant‐based proteins. Eggs contribute to cake volume by stabilizing air bubbles during mixing and baking, and when they are reduced, the cake structure can become more compact. For instance, Xie et al. (2019) found that replacing 10% of the egg with soy protein isolate had no significant effect on cake height, although the specific volume decreased as the level of egg substitution increased. Similarly, Giarola et al. (2022) observed that cakes prepared using sunflower protein meal resulted in a collapsed structure, highlighting the structural limitations that can arise when egg functionality is not sufficiently mimicked.

FIGURE 4.

Images of the produced cake samples C, US10, and US20 represent cake samples formulated with 0% (control), 10%, and 20% replacement of egg yolk with ultrasound‐extracted grape seed protein concentrates, respectively.

TABLE 3.

Physical and chemical properties of the cakes.

Sample	Height (mm)	pH	Moisture (%)	Baking loss (%)	Antioxidant activity (µg Trolox/g)
C	32.45 ± 1.14a	7.75 ± 0.05a	12.65 ± 0.32a	14.70 ± 0.46a	284.48 ± 32.09b
US10	31.93 ± 1.03a	7.82 ± 0.03a	13.33 ± 0.19a	13.81 ± 1.75a	324.19 ± 3.40ab
US20	29.62 ± 0.13b	7.76 ± 0.04a	13.09 ± 0.09a	13.71 ± 0.70a	375.25 ± 9.63a

Note: C represents the control cake formulated with 100% egg yolk, whereas US10 and US20 refer to the cakes in which ultrasound‐modified GSP replaced 10% and 20% of the egg yolk, respectively. Different superscript letters within the same column indicate statistically significant differences among the cake samples (p < 0.05).

The pH values of the cake samples ranged from 7.75 to 7.82, with no statistically significant differences observed among the formulations (p > 0.05). These values fall within the pH range commonly reported for cake products, which has been shown to vary between 5.4 and 7.8 depending on formulation and ingredients (Grossi Bovi Karatay et al. 2022; Nakov et al. ²⁰²⁰). In previous studies, the incorporation of plant‐based ingredients such as grape pomace powder has been associated with a gradual decrease in cake pH as the substitution level increased (Nakov et al. 2020). The stable pH observed in the present study suggests that partial replacement of egg yolk with US‐modified GSP did not substantially alter the acid–base balance of the cake system.

Moisture content is a key quality parameter in cake products, as it directly influences sensory attributes such as softness and freshness, as well as microbial stability during storage. In the present study, the moisture content of the cakes ranged from 12.65% to 13.33%, with no statistically significant differences observed among the formulations (Table 3). These values are consistent with those reported for cakes supplemented with alternative protein sources, such as edible insect flours (Kowalski et al. 2023). Previous studies have shown that cake moisture content can vary widely, typically between 10.66% and 26.7%, depending on factors such as ingredient composition, level of substitution, and baking conditions (Grossi Bovi Karatay et al. 2022; Kowalski et al. ²⁰²³; Levent et al. ²⁰²¹). The relatively consistent moisture levels observed across all samples indicates that partial replacement of egg yolk with US‐modified GSP did not adversely affect moisture retention in the cake matrix.

3.2.2. Baking Loss

The baking loss values showed a slight decreasing trend with the addition of US‐modified GSP, ranging from 13.71% to 14.70%, although no statistically significant differences were observed among the cake formulations (Table 3; p > 0.05). The relatively lower baking loss values may be attributed to the enhanced water‐ and oil‐binding capacities of the US‐derived products (GSP‐UAE), which likely helped retain moisture and fat within the cake matrix during baking. Accordingly, the moisture content was retained better in GSP‐incorporated US10 and US20 cakes (Table 3). Similarly, Díaz‐Ramírez et al. (2016) reported insignificant changes in baking loss values ranging from 15.55% to 16.42% in cakes in which whey protein isolate was used to replace egg up to 50%, while also observing improved moisture retention. Similarly, Grossi Bovi Karatay et al. (2022) reported baking loss values ranging from 8.9% to 9.5% in pound cakes partially substituted with chickpea aquafaba, again with no significant differences among samples. However, complete substitution of egg at 100% has been reported to increase the baking loss (Díaz‐Ramírez et al. ²⁰¹⁶), highlighting partial replacement as a more effective and practical strategy.

3.2.3. Antioxidant Properties

The antioxidant activity of the cake samples clearly increased with the incorporation of GSP, indicating a functional enrichment of the product (Table 3). The control cake (C) exhibited an antioxidant activity of 284.48 ± 32.09 µg Trolox/g, whereas cakes containing US‐modified GSP showed progressively higher values. Antioxidant activity increased to 324.19 ± 3.40 µg Trolox/g in the US10 formulation and further to 375.25 ± 9.63 µg Trolox/g in the US20 cake, demonstrating a dose‐dependent enhancement. This increase reflects the contribution of grape seed–derived bioactive compounds to the antioxidant potential of the final baked product. Among the plant‐based protein sources investigated as egg replacers in bakery products, such as soy, pea, chickpea, sunflower, and hemp proteins, red GSP offers several advantages. First, it is produced from grape seed meal, an abundant agro‐industrial by‐product, supporting the valorization of food processing residues within circular economy frameworks (Singh et al. 2022; Ubaid and Saini ²⁰²⁴). In addition, grape seeds contain bioactive compounds including proanthocyanidins, anthocyanins, flavonols, and phenolic acids, which contribute to their antioxidant activity (Krasteva et al. 2023). A portion of these compounds may be retained during protein extraction, thereby contributing to the antioxidant potential of the resulting protein concentrate. Therefore, unlike many conventional plant protein ingredients that primarily provide nutritional and techno‐functional benefits, GSP may simultaneously enhance both the functional properties and antioxidant capacity of the final product in which it is incorporated. Similar trends have been also reported in previous studies using plant‐based protein or seed‐derived ingredients. For instance, Cingöz (2023) observed that the addition of sour cherry seed protein concentrates to gluten‐free baton cakes significantly increased both total phenolic content and antioxidant capacity, while substitutions up to 5% did not negatively affect cake structure or physical quality. Likewise, Aljobair (2022) demonstrated that incorporating chia seed flour into cake formulations significantly enhanced antioxidant activity through increased free radical scavenging capacity, with optimal sensory acceptance achieved at a 3% substitution level. These studies support the idea that moderate incorporation of bioactive‐rich plant ingredients can improve the nutritional quality of baked goods without compromising product quality.

3.2.4. Textural Properties

Texture is commonly evaluated in cakes using parameters including hardness, cohesiveness, springiness, gumminess, and chewiness (Salehi and Kashaninejad 2018). TPA results indicated that the addition of GSP‐UAE had remarkable effects on such textural properties of the cake samples (Table 4). Hardness represents the force required to compress the foods (Peleg 2019) and is closely related to cake crumb firmness (Figure 5). The hardness values of the control (C) (544.50 ± 4.08 g) and US10 (555.25 ± 44.29 g) cakes were similar, indicating that replacing 10% of egg yolk with GSP‐UAE did not significantly affect cake firmness. In contrast, the US20 cake exhibited a significantly higher hardness value, suggesting the formation of a denser and more compact crumb structure at higher substitution levels (Figure 5). In contrast, cohesiveness, springiness, and resilience showed a slight dose‐dependent decrease, with values ranging from 0.32 to 0.41, 0.78 to 0.81, and 0.11 to 0.15, respectively, although these differences were not statistically significant (Table 4). Cohesiveness reflects the internal strength of the cake structure and its ability to withstand deformation without breaking (Nishinari and Fang 2018). The slight decreasing trend in cohesiveness indicated that partial substitution of egg yolk with GSP did not remarkably weaken the internal structure of the cake. On the other hand, springiness describes the ability of the cake to recover its shape after compression, while resilience reflects how quickly this recovery occurs (Nishinari and Fang 2018). Similar springiness values among the cake samples demonstrate that partial egg yolk replacement did not adversely affect elastic recovery. A slight decrease in resilience was observed in the US20 cake, suggesting a minor reduction in the cake's ability to rapidly regain its structure after deformation. These findings are consistent with earlier studies reporting that the addition of plant‐based proteins to cake formulations increases hardness (Majzoobi et al. 2014) and decreases cohesiveness and springiness (Mohamed et al. 2025). This can be considered a characteristic outcome of integrating functional proteins, such as grape seed protein, into the cake matrix. The fact to obtaining a firmer final product could be due to the enhanced gelling behavior and formation of a more rigid protein network (Yazici and Ozer 2021), together with a reduction in free water caused by the water‐binding capacity of plant proteins (Majzoobi et al. 2014). Gumminess and chewiness are secondary texture parameters derived from hardness, cohesiveness, and springiness (Peleg 2019) and represent the energy required to disintegrate and chew the cake. No significant differences were observed among the samples for either parameter. These findings indicate that partial substitution of egg yolk with GSP‐UAE did not negatively affect the overall chewing characteristics of the cakes. Overall, the results demonstrate that substitution level plays a critical role in determining cake texture. Incorporation of GSP‐UAE at 10% maintained textural properties comparable to the control cake, while higher substitution (20%) led to increased firmness and slightly reduced elasticity.

TABLE 4.

Textural properties of the cakes.

Sample	Hardness (g)	Cohesiveness	Springiness	Gumminess (g)	Chewiness index (g)	Resilience
C	544.50 ± 4.08b	0.41 ± 0.06a	0.81 ± 0.02a	225.30 ± 34.50a	183.15 ± 32.45a	0.15 ± 0.02a
US10	555.25 ± 44.29b	0.36 ± 0.07a	0.79 ± 0.03a	234.13 ± 30.18a	189.83 ± 26.88a	0.14 ± 0.04a
US20	749.50 ± 30.21a	0.32 ± 0.02a	0.78 ± 0.02a	237.45 ± 0.15a	184.00 ± 3.70a	0.11 ± 0.02a

Note: C represents the control cake formulated with 100% egg yolk, whereas US10 and US20 refer to the cakes in which ultrasound‐modified GSP replaced 10% and 20% of the egg yolk, respectively. Different superscript letters within the same column indicate statistically significant differences among the cake samples (p < 0.05).

FIGURE 5.

Texture profiles of the cake samples C, US10, and US20 represent cake samples formulated with 0% (control), 10%, and 20% replacement of egg yolk with ultrasound‐extracted grape seed protein concentrate, respectively.

Egg yolk is a multifunctional ingredient in cake formulations, contributing simultaneously to emulsification, aeration, moisture retention, texture development, and structure stabilization due to its protein composition (Deleu, Melis, et al. ²⁰¹⁷; Pycarelle et al. ²⁰²¹). During batter mixing, egg yolk lipoproteins stabilize the oil–water and air–liquid interfaces, promoting the incorporation and retention of air bubbles that are essential for cake volume and crumb development (Pycarelle et al. 2021). During baking, egg proteins form a three‐dimensional network through disulfide bonds and hydrophobic interactions (Deleu, Wilderjans, et al. ²⁰¹⁷), contributing to the final texture, elasticity, and structural integrity of the cake. Consequently, the replacement of egg yolk remains a significant challenge because alternative ingredients must simultaneously provide several technological functions rather than a single property. Although proteins derived from other plant sources have been investigated as partial egg replacers due to their emulsifying, water‐binding, and gelling capacities, plant proteins generally exhibit lower functionality than egg proteins and often require formulation adjustments, protein blending, or the addition of emulsifiers to achieve comparable cake quality (Jarpa‐Parra et al. ²⁰¹⁷). For example, lentil proteins may increase cake hardness and chewiness with lower baking loss (Jarpa‐Parra et al. ²⁰¹⁷), while soy proteins can produce cakes with acceptable volume and texture when combined with suitable emulsifying systems (Lin et al. 2017). In the present study, the enhanced water‐ and oil‐binding capacities, combined with the structural modifications revealed by FTIR and SEM, likely enabled GSP‐UAE to partially perform some of the technological functions normally provided by egg yolk in cake formulations. This interpretation is supported by the comparable quality attributes observed between the control and US10 cakes, indicating that moderate substitution levels can be achieved without substantially compromising cake quality.

3.2.5. Color Properties

Color is a key quality attribute of bakery products, including cakes, and plays an important role in consumer acceptance. The external and internal surface color parameters of cakes are shown in Table 5. The incorporation of GSP to the cake formulation significantly contributed to the color changes in the final product (Figure 4). The lightness (L*) values of cakes statistically decreased (p < 0.05) depending on the increasing amount of GSP. These results can be attributed to the Maillard reaction occurring during the baking due to amino acids and sugar constituents in the cake formulation (Yalcin 2025). Examining the a* and b* values of cakes, it was observed that a tendency to redness and blueness is linked to an increase in GSP ratio associated with it containing purple‐blue anthocyanin. The control cake exhibited higher yellowness (b*) due to including carotene and xanthophyll pigments derived from egg yolk. These findings were in line with the previous studies (Nakov et al. 2020; Yalcin ²⁰²⁵).

TABLE 5.

Internal and external color properties of the cakes.

Surface	Sample	L*	a*	b*	ΔE
Internal	C	63.96 ± 1.49a	−1.46 ± 0.08c	24.86 ± 0.57a	—
	US10	47.65 ± 2.18b	5.39 ± 0.37b	18.73 ± 0.62b	18.72
	US20	42.64 ± 1.60c	7.62 ± 0.29a	18.42 ± 0.62b	24.05
External	C	61.55 ± 0.45x	15.42 ± 0.52x	40.31 ± 0.68x	—
	US10	54.68 ± 2.81y	13.14 ± 1.65y	31.14 ± 0.69y	10.65
	US20	47.91 ± 1.96z	12.74 ± 0.52y	22.98 ± 1.65z	21.05

Note: C represents the control cake formulated with 100% egg yolk, whereas US10 and US20 refer to the cakes in which ultrasound‐modified GSP replaced 10% and 20% of the egg yolk, respectively. Different superscript letters (a–b) within the same column indicate statistically significant differences in the internal color of cake samples, while different superscript letters (x–z) indicate significant differences in external color (p < 0.05).

4. Conclusion

This study demonstrated that red grape seed meal can be successfully valorized into a functional protein concentrate through UAE. Compared to conventional AE, US treatment enhanced protein recovery, increased soluble protein content fourfold, and improved key techno‐functional properties such as water‐ and oil‐binding capacities. SDS–PAGE, FTIR, and SEM results collectively demonstrated that UAE improved protein recovery and extractability without causing substantial protein degradation. The enhanced band intensity, slight conformational changes, and more porous particle structure observed in GSP‐UAE likely contributed to its superior water‐ and oil‐binding capacities and its improved performance as a functional ingredient in cake formulations. Cakes formulated with 10% egg yolk replacement exhibited physical and textural properties comparable to the control cake. Higher substitution (20%) led to increased firmness and a denser crumb structure, highlighting the importance of optimizing replacement levels to balance functionality and product quality. In addition to its technological performance, the incorporation of grape seed protein significantly enhanced the antioxidant capacity of the cakes, reflecting the contribution of grape seed‐derived phenolic compounds. This finding highlights the dual functionality of GSP, which not only serves as a protein ingredient but also contributes antioxidant activity, unlike many conventional plant‐based protein concentrates that are primarily used for their nutritional and techno‐functional properties. Consequently, antioxidant activity increased in a dose‐dependent manner, reaching 375.25 µg Trolox/g in the US20 formulation. Overall, the partial egg yolk replacement using US‐modified grape seed protein offers a viable approach to develop value‐added cakes with enhanced properties while contributing to the sustainable utilization of grape processing by‐products as alternative plant‐based ingredients in bakery products. Future work should address the scalability and economic feasibility of US‐assisted extraction for industrial production of GSPs. To fully explore its potential, consumer‐based sensory studies with a diverse panel are needed to better assess market acceptance of cakes formulated with grape seed protein. Further investigations should evaluate protein digestibility, the bioaccessibility of phenolic compounds after baking, and the detailed polyphenol profile of protein concentrates obtained by different extraction methods. Such studies would provide deeper insight into the retention and distribution of bioactive compounds, the role of protein–phenolic interactions, and their contribution to the functional, antioxidant, and technological properties of the resulting ingredients.

Author Contributions

Yaren Didem Esendemir: methodology, investigation, data curation, visualization, formal analysis, writing – original draft. Marıam Alradwan: methodology, investigation, data curation, visualization, writing – original draft. Semanur Yildiz: conceptualization, methodology, software, data curation, formal analysis, resources, writing – original draft, writing – review and editing, supervision, funding acquisition, visualization. İrem Onay: investigation. Senanur Kiziltaş: investigation, funding acquisition. Tuğba Karadeniz: investigation.

Conflicts of Interest

The authors declare no conflicts of interest.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.