Optimizing Chickpea Cooking Water (Aquafaba): Enhancing Superior Foaming and Emulsifying Properties Through Concentration Protocols

Abstract

Aquafaba, the viscous liquid obtained from cooking chickpeas, has gained significant attention in the food industry due to its remarkable foaming and emulsifying properties, positioning it as a promising plant-based alternative to egg whites. This study investigated the effects of reheating on aquafaba’s functional properties, with a focus on its compositional concentration and molecular structural changes. Reheating was found to enhance both foaming and emulsifying capacities, with the most favorable results observed when the remaining liquid ratio was adjusted to 70–50%. Detailed molecular size analysis identified proteins and carbohydrates in the 30–100 kDa range as critical contributors to foam formation and stability. Furthermore, enzymatic treatments revealed that the synergistic interactions between proteins, pectins, and carbohydrates are key to aquafaba’s multifunctionality, enabling it to replicate the desirable properties of egg whites in various food applications. These findings not only advance our understanding of aquafaba’s molecular mechanisms but also demonstrate the potential of reheating as a practical strategy to optimize its properties for a wider range of culinary and industrial uses. This study underscores aquafaba’s versatility and highlights its role as a sustainable, plant-based ingredient capable of meeting the growing demand for vegan and allergen-free food products.

1. Introduction

The growing demand for sustainable ingredients has led the food industry to explore plant-based alternatives to animal-derived proteins. Aquafaba, derived from the Latin words “aqua” (water) and “faba” (bean), is the cooking water from legumes, particularly chickpeas (Cicer arietinum L.), and has gained popularity as a substitute for egg whites in sauces, foams, and baked goods due to its foaming and emulsifying properties [1,2,3]. As a novel natural food additive, aquafaba is expected to boost demand for legumes and reduce wastewater generated from bean processing [3].

Chickpea, an annual herbaceous legume, is a rich source of proteins, carbohydrates (primarily starch and fiber), minerals, and vitamins. Its protein composition includes 8–12% albumin, 53–60% globulin, 19–24% glutelin, and 3–6% prolamin [4]. Soaking and cooking chickpeas facilitate water absorption, leading to protein hydration, denaturation, starch gelatinization, solubilization, and depolymerization [3,5]. Prolonged cooking causes the starch to expand and gelatinize, which disrupts the seed coat due to internal pressure. Consequently, the leached components in the cooking water include not only proteins and sugars but also undissolved components such as starch [6,7,8]. In other words, various components elute into the boiling water during cooking.

When preparing an emulsion with a protein acting as a surfactant, the protein reduces the interfacial tension by adsorbing onto the surface of dispersed particles, thereby stabilizing the emulsion by forming a robust adsorption film [9,10]. Surfactants commonly found in food include casein and whey protein from milk, lecithin and globulin from soybeans, and lecithin and ovalbumin from eggs, all of which are known for their foaming and emulsifying properties [11]. Aquafaba proteins primarily consist of low-molecular-weight species (≤25 kDa), likely albumins, which are effective in forming stable foams [1,12]. However, aquafaba’s protein content is relatively low, at approximately 1.5% [1,13]. Therefore, the combination of low-molecular-weight protein and carbohydrates likely confers the foaming and emulsifying properties seen in aquafaba. Additionally, components such as cellulose, pectin, and gelatinized starch may enhance these properties by increasing the viscosity and providing structural support [14,15].

Recent studies have provided insights into the functional properties and applications of aquafaba. For example, Aslan et al. optimized foam drying techniques to enhance the storage stability and usability of aquafaba in powdered forms [16]. Echeverria-Jaramillo et al. demonstrated the sustainability and functional potential of aquafaba derived from various soybean varieties [17]. These findings underscore the growing interest in aquafaba as a plant-based functional ingredient, particularly for its foaming and emulsifying properties. Methods to improve aquafaba’s functionality, such as extending the boiling time or reducing the water content to concentrate leached components, have also been reported [14,18]. However, because aquafaba is a byproduct of bean cooking, excessive focus on its quality may adversely affect the nutritional and sensory properties of the cooked beans. To address this challenge, in this study, we aimed to develop a method that could enhance aquafaba’s foaming and emulsifying properties without compromising the quality of the cooked beans. Specifically, we examined the effects of concentration through reheating and analyzed the contributions of specific components using enzymatic treatments and molecular size analysis.

2. Materials and Methods

2.1. Materials

Dried chickpeas were purchased from a specialty bean and grain store (Suzuya, Miyagi, Japan). Beans with visible blemishes were removed prior to use. Fresh eggs were purchased from a grocery store and stored at 4 °C until use. All enzymes were purchased from Amano Enzymes (Nagoya, Japan). Samoase PC10F (protease) with an activity of ≥90,000 U/g, pectinase G “Amano” (pectinase) with an activity of ≥1200 U/g, and gluczyme NL4.2 (glucoamylase) with an activity of ≥1200 U/g were used in this study.

2.2. Aquafaba Preparation

The cooking method for the chickpeas was partially modified from that described by Stantiall et al. [10]. Dry chickpea seeds (~200 g) were washed and rehydrated by soaking in distilled water at a ratio of 1:4 (w/w) for 16 h at 4 °C. The soaked chickpeas were cooked in boiling water at a 3:1 wt ratio (water/chickpeas) for 40 min. After cooking, the water and chickpeas were transferred to a glass bowl and stored at 4 °C for 24 h. Then, the aquafaba was separated from the cooked grains using a strainer. The average hardness of the chickpeas after cooking was 3.53 N, which is suitable for consumption [19]. The samples were categorized as follows: control: standard aquafaba preparation, and concentrated: aquafaba with reduced water content achieved through evaporation (see Section 2.3). These categorizations allowed for the investigation of the effects of aquafaba concentration on its foaming and emulsifying properties.

2.3. Concentration of Aquafaba

The effects of concentration on aquafaba’s foaming and emulsifying properties were explored by varying the remaining liquid ratio. The process was divided into eight levels as follows: 100%, 90%, 80%, 70%, 60%, 50%, 40%, and 30% (w/w). These levels represent the percentage of the remaining liquid relative to the original weight of aquafaba before concentration, with 100% referring to the original, unconcentrated aquafaba. To achieve the desired concentration level, 50 mL of aquafaba was heated in a glass beaker with continuous stirring using a magnetic stirrer until the target remaining liquid ratio was reached (e.g., 90% or 80%).

2.4. Color

The color intensity was measured using a spectrophotometer (CM-700d, KONICA MINOLTA, Tokyo, Japan) using L*, a*, and b* values, according to the CIE color scale. L* represents brightness from 0 (black) to 100 (white). The other two coordinates represent redness (+a*) to greenness (–a*) and yellowness (+b*) to blueness (–b*). The color change (ΔE) was determined to compare the color of the reheated aquafaba with that of the untreated aquafaba. All experiments were performed in triplicate.

2.5. Viscosity

The viscosity of the aquafaba was measured using a rotational viscometer (VISCO; Atago Co., Ltd., Tokyo, Japan). Aquafaba was dispensed in a glass beaker (15 mL; Atago Co., Ltd.). Measurements were conducted under spindle A2 conditions, a rotational speed of 150 rpm, and sample temperatures of 4 °C, 25 °C, and 40 °C, with data collected 1 min after the spindle rotational speed was stabilized. The sample temperature during viscosity measurement was controlled by a Temp Controller 6900 (Atago Co., Ltd.) attached to the viscometer.

2.6. Foaming Properties

The foaming properties were evaluated by measuring foaming capacity and foam stability. Foam capacity was assessed in terms of overrun, which represents the expansion rate of the whipped foam and was calculated by measuring the foam’s volume. Foam stability was evaluated by assessing the amount of liquid drainage and foam collapse after leaving the foam at 5 °C for 16 h. Aquafaba samples (30 mL) were pre-equilibrated to the initial target temperature (4 °C, 25 °C, or 40 °C) in a refrigerator or water bath prior to the whipping process. The samples were then placed in a container and whipped for 2 min at a speed of 1050 rpm using a hand mixer (Dretec Co., Ltd., HM-702, Saitama, Japan). During the whipping process, the temperature was not actively controlled. After whipping, the foam was carefully transferred into a 300 mL measuring cup to determine its volume. The percentage of foam expansion was calculated using the following equation by Echeverria-Jaramillo et al. [17]:

Foam overrun (%) = (V_foam − V_liquid)/V_liquid × 100

(1)

where V_liquid is the initial volume of the aquafaba before whipping, and V_foam is the volume of the whipped foam measured in a 300 mL measuring cup after whipping.

To quantify foam stability, the foam was carefully transferred to a plastic funnel placed on top of a 50 mL graduated cylinder immediately after the foaming process. The weight of the retained foam and the drained liquid was recorded after leaving it at 5 °C for 16 h. The retained foam weight was calculated using Equation (2), as follows:

Liquid ratio in foam (%) = (V_li − V_lt)/(V_li − V_l0) × 100

(2)

where V_l0 is the liquid volume at time 0, V_lt is the liquid volume after 16 h, and V_li is the initial sample volume (30 mL). Analyses of the foaming ability and foam stability were performed in triplicate.

2.7. Emulsifying Properties

The emulsifying properties were assessed following the methodology of Mustafa et al. [1], with partial adaptations. Emulsions were prepared by mixing 15 mL of untreated or concentrated aquafaba and 15 mL of canola oil (Nissin Oil Mills, Tokyo, Japan), for 1 min, in a homogenizer (Bio-Gen PRO200, PRO Scientific Inc., Oxford, CT, USA) at 5000 rpm. Subsequently, the resultant emulsion was quantitatively transferred into a 100 mL graduated cylinder and stored at 25 °C (room temperature). The emulsion stability was evaluated by measuring the amount of liquid separated from the emulsion after 7 days. Results represent the averages of three independent replicates.

The emulsifying capacity was calculated using Equation (3), as follows:

Emulsifying capacity (%) = V_et/V_e0 × 100

(3)

where V_e0 is the emulsion volume at time 0, and V_et is the volume of the emulsion after time t.

For comparative purposes, the emulsifying capacity of the egg yolk at 5 °C was also ascertained, given its extensive use in emulsions such as mayonnaises.

2.8. Proximate Analysis

The general nutritional ingredients of chickpeas, cooked chickpeas, aquafabas, and concentrated aquafabas were analyzed. Moisture, crude protein, crude fat, ash, and crude fiber contents were determined according to the standard methods described by the AOAC [20]. Moisture content was measured using an atmospheric pressure drying method (135 °C). The crude protein content was determined using the Kjeldahl method; the nitrogen-to-protein conversion factor was 6.25. The crude fat content was determined using chloroform-methanol extraction. Dietary fiber content was determined using the Prosky method. The carbohydrate content was calculated using Equation (4), as follows (Monro and Burlingame [21]):

Total carbohydrate (g/100 g) = 100 − [moisture (%) + crude protein (%) + crude fat (%) + ash (%)]

(4)

The sodium content was measured using atomic absorption spectroscopy.

2.9. Enzyme Treatment

The objective of the enzyme treatment was to identify which components of aquafaba—proteins or carbohydrates—contribute to its foaming and emulsifying properties. To achieve this, the following three enzymes with distinct functions were used: protease to target proteins, pectinase to degrade pectin, and glucoamylase to hydrolyze non-pectin carbohydrates. The enzymes were heat-inactivated by incubating the samples at 95 °C for 3 min prior to analysis to prevent any enzymatic activity from interfering with the results. Two test conditions were employed as follows:

• Test 1: Aquafaba was treated with enzymes under optimal conditions to evaluate their effects. The aquafaba was prepared at the optimal pH for each enzyme (protease: pH 7.5; pectinase: pH 4.5; glucoamylase: pH 4.5), mixed with 1% enzyme per dry matter of aquafaba, and adjusted to the optimum temperature (protease: 55 °C; pectinase: 45 °C; glucoamylase: 55 °C) for 2 h.
• Test 2: This control condition was designed to isolate the effects of enzymatic activity. To achieve this, aquafaba was adjusted to the same pH and temperature conditions as in Test 1 but without the addition of enzymes.

The foaming and emulsifying properties were evaluated for both enzyme-treated (Test 1) and non-treated (Test 2) samples, enabling a direct comparison to determine the specific contributions of the enzymatic treatment.

2.10. Molecular Size Analysis and Qualitative Testing of Proteins and Carbohydrates

The remaining liquid ratio of 70% aquafaba was centrifuged at 13,000 rpm for 10 min at 25 °C and separated into supernatant and precipitate; 0.5 mL of ion-exchanged water was added to the precipitate obtained from 1 mL of aquafaba and used for testing the bubbling properties. Then, 0.5 mL of the supernatant was filtered through a 100 kDa ultrafiltration filter and centrifuged at 10,000 rpm for 10 min at 25 °C to obtain the eluate. Subsequently, the eluate was filtered through 50 kDa, 30 kD, and 10 kDa ultrafiltration filters in sequence under the same conditions. The Amicon Ultra 0.5 mL ultrafiltration filters (Merck Millipore Ltd., Tullagreen, Ireland) were used throughout the process. Then, 0.5 mL of these final eluates was placed in a 2 mL tube, stirred vigorously for 1 min, and then allowed to stand at room temperature (25 °C). The height of the bubbles was measured every time, and their respective volumes were calculated.

Protein identification was performed using ninhydrin. To 100 μL of each fraction, 100 μL of a 2% ninhydrin solution (FUJIFILM, Tokyo, Japan) was added and mixed. The mixture was then heated at 100 °C for 5 min, and the resulting solution was examined for blue coloration.

Carbohydrate identification was performed using anthrone-sulfuric acid. To 100 μL of each fraction, 100 μL of a 10 mmol/L anthrone-sulfuric acid solution (FUJIFILM) was added and mixed. The mixture was then heated at 100 °C for 10 min, and the resulting solution was examined for blue coloration.

2.11. Statistics

All data are presented as the mean ± SD. Significant differences in values were analyzed via one-way analysis of variance using SPSS statistical software (version. 27.0, IBM, Armonk, NY, USA). Tukey’s and Bonferroni’s methods were used for multiple comparisons, with the statistical significance level set at p < 0.05. All measurements were performed at least in triplicate.

3. Results and Discussion

3.1. Foaming Properties Depend on Whipping Temperature

Temperature significantly influences the foaming properties of proteins, making it essential to investigate its impact on aquafaba. As shown in Figure 1, the foaming ability across all temperatures was exceptionally high, with overruns ranging from 700% to 800%, and no significant differences were observed. However, the drainage volume revealed notable temperature-dependent variations. At 4 °C, 68.1% of the foam liquid drained, whereas at 25 °C and 40 °C, drainage was significantly reduced to approximately 40%. These results indicate that although aquafaba exhibits excellent foaming ability at both high and low temperatures, whipping at room temperature or around 40 °C results in more stable foams compared to whipping at lower temperatures.

In contrast to egg whites, which are commonly whipped at low temperatures to enhance foam stability, aquafaba demonstrates superior stability at higher temperatures. For egg whites, the foaming process benefits from increased temperatures, as reduced viscosity and surface tension promote foam formation, but the resulting foams tend to collapse more easily due to the weakened protein structures [22]. This is primarily attributed to ovoglobulin and ovomucin, which form thin, stabilizing films around air bubbles under cold conditions. However, our findings suggest that the factors contributing to aquafaba’s foam stability differ from those in egg whites. The non-protein components in aquafaba, such as polysaccharides and pectins, may play a critical role in stabilizing the foam by increasing viscosity and elasticity, thus preventing bubble collapse [23]. Additionally, plant-derived proteins and surface-active compounds like saponins might further enhance the foaming properties of aquafaba, with their effects potentially optimized around 40 °C [24].

These findings highlight the importance of non-protein components in the foaming stability of aquafaba and the significant role of temperature in modulating their physicochemical properties. Consequently, all subsequent foaming experiments in this study were conducted at 25 °C to ensure consistent and optimal conditions were maintained.

3.2. Enhanced Foaming and Emulsifying Properties by Concentration

We investigated the effect of aquafaba concentration on its foaming and emulsifying properties. Lower remaining liquid ratios correspond to higher concentrations of solid components due to reheating. As shown in Figure 2, the foaming ability of reheated aquafaba, except for the 30% concentration, exceeded that of egg whites at all remaining liquid ratios. However, foaming ability decreased as concentration increased, exhibiting an inverse relationship with the remaining liquid ratio. Foam stability, evaluated by drainage volume after 24 h, was consistently superior in aquafaba compared to that in egg whites across all ratios. Notably, the 80–50% range exhibited the least drainage, suggesting this concentration range is optimal for foam stability. Emulsifying properties were also enhanced across all the remaining liquid ratios, with emulsification values reaching 100% at concentrations of 70%, 60%, and 50%. Emulsions in these concentrations remained stable without phase separation, even after one week. These results indicate that reheating significantly enhances the functional properties of aquafaba, particularly within the 70–50% concentration range. However, we evaluated emulsion stability based on macroscopic observations and liquid drainage measurements. Although this method provides useful insights into macroscopic behavior, it may not capture the finer details, such as changes in droplet size distribution. Advanced methods, including light scattering analysis, could provide a more comprehensive understanding of emulsion stability and are recommended for future studies.

The observed improvements in foaming and emulsifying properties can be attributed to structural changes in aquafaba components induced by reheating. Protein denaturation during reheating likely exposes hydrophobic regions, increasing surface activity and facilitating the formation of stable foam and emulsification films [25]. This finding is consistent with those of a previous study on thermal concentration methods, which reported enhanced foaming and emulsifying properties due to the denaturation and aggregation of proteins under heat treatment [26]. Simultaneously, aggregated proteins may form dense interfacial films, further stabilizing the structure. Polysaccharides, known for their viscosity and gel-forming capabilities, likely contribute to foam and emulsion stability by interacting with proteins and reinforcing the structural integrity of these systems [3,27].

The concentration process through reheating increased the functional components in aquafaba, leading to improved foam stability and reduced phase separation in emulsions. Although these findings demonstrate that the concentration process enhanced aquafaba’s foaming and emulsifying properties, the underlying mechanisms remain to be fully elucidated. The observed improvements were likely due to the increased availability of solid components at the interface. However, further studies are needed to confirm the specific role of protein–polysaccharide interactions in these functional properties.

3.3. Color Value After Concentration

Concentration-induced changes in the color and viscosity of aquafaba were analyzed to assess the impact of chemical reactions and component interactions on its physical properties. As shown in

Table 1. Influence of concentration on aquafaba color and viscosity depending on concentration levels.

		Aquafaba Concentration Level (%)
		100	90	80	70	60	50	40	30
Color	L*	72.8 ± 0.7 bc	69.1 ± 0.2 c	84.7 ± 0.4 a	78.6 ± 0.8 abc	79.9 ± 0.4 ab	55.1 ± 13.7 d	38.9 ± 11.8 e	28.6 ± 0.1 e
	a*	3.3 ± 0.1 e	4.3 ± 0.0 d	1.2 ± 0.0 h	2.7 ± 0.1 f	2.0 ± 0.0 g	5.5 ± 0.6 c	8.0 ± 0.4 b	8.9 ± 0.0 a
	b*	23.4 ± 0.1 cd	27.0 ± 0.1 b	21.5 ± 0.1 d	25.6 ± 0.1 bc	26.0 ± 0.0 bc	30.2 ± 3.0 a	31.0 ± 4.1 a	30.1 ± 0.1 a
	⊿E		5.6 ± 0.1	11.7 ± 0.4	5.8 ± 0.7	7.2 ± 0.4	20.5 ± 12.0	36.0 ± 10.7	45.6 ± 0.1
Visosity	4 °C	23.2 ± 1.5 f	32.7 ± 1.9 ef	40.4 ± 3.3 de	55.2 ± 1.1 d	93.1 ± 2.0 c	106 ± 8.2 c	174 ± 9.2 b	259 ± 31.3 a
(mPa·s)	25 °C	18.6 ± 1.9 f	23.9 ± 2.5 ef	31.6 ± 2.3 e	28.7 ± 1.67 ef	59.7 ± 2.2 d	75.7 ± 3.6 c	132 ± 3.4 b	233 ± 12.2 a
	40 °C	19.7 ± 0.7 e	23.2 ± 0.4 e	23.1 ± 0.8 e	23.9 ± 0.2 e	46.1 ± 1.2 d	56.6 ± 0.4 c	107 ± 2.2 b	186 ± 10.0 a

Significant differences are indicated by different lowercase letters in accordance with Tukey’s test. p < 0.05, n = 6.

, the L* value (lightness) decreased with an increase concentration, whereas the a* (redness) and b* (yellowness) values increased. This indicates that the concentration process through reheating gradually transformed aquafaba from a yellowish, transparent liquid into a brownish liquid. These color changes were primarily attributed to the Maillard reaction between proteins and reducing sugars, which accelerates under higher temperatures or a longer cooking time [27]. In the present study, the lower the remaining liquid ratio, the longer the cooking time, which likely resulted in more pronounced color changes. Additionally, the increase in the number of solids in the solution due to the concentration process could have influenced the observed changes by altering light scattering properties. This suggests that both the Maillard reaction and the increased solid content during concentration contribute to the browning of aquafaba.

3.4. Viscosity After Concentration

The viscosity of aquafaba was analyzed to evaluate the effects of the concentration process through reheating on its physical properties. Viscosity varied with temperature and concentration. At the same remaining liquid ratio, viscosity was highest at 5 °C and lowest at 40 °C, likely due to reduced molecular motion and stronger intermolecular interactions at lower temperatures. As the concentration increased, viscosity also rose, with aquafaba boiled down to 30% forming a gel-like consistency. This thickening phenomenon can be attributed to the accumulation of Maillard reaction products and the aggregation of proteins and polysaccharides, which increase molecular weight and enhance solution viscosity [28]. High molecular weight Maillard reaction products also exhibit dietary fiber-like properties, contributing to gelation and viscosity enhancement [27]. Prior to concentration, the viscosity of aquafaba was approximately one-fifth that of egg whites. To achieve a viscosity comparable to that of egg whites, aquafaba required a concentration of 40–50%. However, as shown in Figure 1 and Figure 2, sufficient foam formation was achieved even before that concentration was reached. This suggests that the interactions among aquafaba components, rather than viscosity alone, play a critical role in its foaming properties [23,29].

The concentration process has been shown to influence aquafaba’s functionality by altering its physical properties, such as color and viscosity. As discussed in Section 3.2, protein denaturation and Maillard reaction products contribute significantly to the stability of foams and emulsions. Additionally, polysaccharides, which are abundant in aquafaba, play a key role in viscosity enhancement and gel formation, further supporting foam and emulsion stability [3]. The changes in color and viscosity observed during reheating, therefore, reflect these underlying molecular interactions and their impact on aquafaba’s overall functionality.

3.5. Changes in the Composition of Aquafaba Due to Concentration

Table 2. Chemical composition of raw chickpea, cooked chickpea, and chickpea cooking water; concentrated aquafaba 100%, 70%, and 50%.

Ingredient	Chickpea		Concentrated Aquafaba
	Dry	Cooked	100%	70%	50%
Water content (g/100 g)	12.2	67.4	98.3	95.2	94.1
Protein (g/100 g dw)	25.5	24.8	41.2	22.9	25.4
Carbohydrate (g/100 g dw)	78.9	66.0	41.2	56.3	64.4
Sugars (g/100 g dw)	53.6	58.0	17.6	45.8	52.5
Dietary fiber (g/100 g dw)	21.1	29.1	23.5	10.4	11.9
Crude fat (g/100 g dw)	6.5	7.4	<dl	<dl	<dl
Ash (g/100 g dw)	3.1	1.8	17.6	10.4	10.2
Energy (kcal/100 g)	420	313	294	292	339
Sodium (mg/100 g)	2	0	59	42	34

presents the major components of dry chickpeas, cooked chickpeas, and aquafaba concentrated to 100%, 70%, and 50% remaining liquid ratios, analyzed on a dry-weight basis. Concentration-induced changes in aquafaba composition are of particular interest, as they contribute to its enhanced functionality. The concentration process through reheating increased the overall solid content, particularly that of carbohydrates.

Dry chickpeas contained approximately 78.9% carbohydrates and 25.5% protein, with the primary carbohydrate being sugars (53.6%), mostly in the form of starch. These values are consistent with those reported previously [12]. Cooked chickpeas showed a slight decrease in carbohydrate content (66.0%) but retained a similar protein level (24.8%), indicating a minimal loss of protein during cooking. In aquafaba, before reheating, the total solid content was 1.7% (w/w), consisting primarily of 0.7% protein and 0.7% carbohydrates, with no detectable fat. These values align with the commonly reported range of 1–5% solids for aquafaba [10,14]. Converted to a dry-weight basis, the carbohydrate and protein content of 100% aquafaba corresponded to 41.2 g/100 g dw each. The carbohydrate content increased disproportionately during the concentration process, increasing from 41.2 g/100 g dw at 100% to 56.3 g/100 g dw at 70% and 64.4 g/100 g dw at 50%. Sugars, which accounted for 17.6 g/100 g dw in 100% aquafaba, increased to 45.8 g/100 g dw at 70% and 52.5 g/100 g dw at 50%. This disproportionate increase may have resulted from the partial hydrolysis of polysaccharides into simpler sugars, as well as the potential crystallization or aggregation of sugars during concentration [30]. Protein content, however, showed a less consistent trend, decreasing from 41.2 g/100 g dw in 100% aquafaba to 22.9 g/100 g dw in the 70% concentration, before increasing slightly to 25.4 g/100 g dw at 50%. This variation may be attributed to changes in protein solubility and aggregation during concentration.

The exceptional foaming and emulsifying properties of aquafaba indicate that even relatively small amounts of eluted components can significantly contribute to its functionality. These results suggest that concentration enhances the carbohydrate content of aquafaba, which plays a significant role in its improved functionality. The accumulation of Maillard reaction products, including both low and high molecular weight compounds, may also contribute to these changes. These compositional changes highlight the importance of carbohydrates and their interactions with proteins and other components in the functional properties of concentrated aquafaba. Further studies on the molecular mechanisms underlying these changes will provide deeper insights into aquafaba’s potential as a functional food ingredient.

3.6. Influence of Enzyme Treatment on the Foaming and Emulsifying Properties of Aquafaba

To investigate the components responsible for the foaming and emulsifying properties of reheated aquafaba, we treated it with the following three enzymes: protease, pectinase, and glucoamylase. These enzymes were used to hydrolyze proteins, pectin, and non-pectin carbohydrates, respectively. A control sample without enzymes was prepared under the same pH and temperature conditions as the enzyme-treated samples.

As shown in Figure 3, in the control samples, foams were formed and remained stable for 90 min under all conditions. Notably, the foam volume was higher in the pectinase and glucoamylase controls than in the protease control, likely because the pectinase and glucoamylase controls were whipped at pH 4.5, whereas the protease control was whipped at pH 7.5. A lower pH reportedly improves foaming properties [31]. In contrast, no foams were observed in the enzyme-treated samples, regardless of the enzyme used. These results indicate that proteins, pectin, and non-pectin carbohydrates are all essential for the foaming properties of aquafaba, and the degradation of any of these components prevents foam formation. Foam stability is generally enhanced by the adsorption of proteins onto bubble surfaces, forming stabilizing films. However, the small amount of protein in aquafaba would not be sufficient to independently form stable foams, as the foam would likely collapse before a robust film could form. This suggests that interactions between proteins and polysaccharides play a critical role in improving foaming properties and foam stability. Proteins primarily contribute to foam formation through their surface activity, while polysaccharides stabilize the foam by enhancing viscosity and reinforcing the structure [10]. Thus, the foaming properties and stability of aquafaba are likely dependent on the synergistic interactions between these components.

As shown in Figure 4, the emulsifying properties followed a similar trend. In the control samples, emulsions remained stable for 90 min. When treated with pectinase or glucoamylase, emulsions initially formed but gradually separated over time, with approximately 80% of the emulsified liquid remaining after 90 min. In contrast, protease treatment caused emulsions to separate rapidly, with only 58% of the emulsified liquid remaining after 10 min. These results suggest that while pectin and non-pectin carbohydrates contribute to emulsion stability, proteins play a more dominant role in the emulsification process.

Overall, the findings demonstrate that proteins, pectin, and non-pectin carbohydrates collectively contribute to the foaming and emulsifying properties of aquafaba, with proteins playing a particularly critical role in ensuring emulsion stability.

3.7. Molecular Weight-Dependent Foaming Properties of Reheated Aquafaba

The experiment described in Section 3.5 has demonstrated that proteins and carbohydrates contribute to the foaming properties of reheated aquafaba eluates. Based on these findings, this study investigated the foaming components of reheated aquafaba with a focus on molecular size. For this purpose, aquafaba with a remaining liquid ratio of 70% was used. As the solution was turbid, insoluble substances (precipitates) were first removed via centrifugation. Neither foaming nor foam stability were observed in the precipitate, suggesting that the foaming components are present in the soluble fraction. To further investigate this, the soluble components were separated using a 100 kDa ultrafiltration filter. A small amount of foaming was observed in the fraction above 100 kDa, whereas the fraction below 100 kDa showed stronger foaming activity. The soluble fraction below 100 kDa was further divided into four molecular weight ranges, 50–100 kDa, 30–50 kDa, 10–30 kDa, and below 10 kD, using ultrafiltration filters of corresponding pore sizes. The results are summarized in

Table 3. Foam volume (cm³) of each fraction after the ultrafiltration and qualitative analysis of proteins and carbohydrates.

Fraction	Time (h)						Ninhydrin	Anthrone
	0	1	2	3	6	24
AF	1.10	1.10	1.10	0.94	0.94	0.71	++	++
AF-S	1.18	1.18	1.18	1.18	1.18	0.31	++	++
AF-P	0.08	0.00	0.00	0.00	0.00	0.00	±	+
>100 kDa	0.47	0.31	0.24	0.24	0.24	0.08	−	±
50–100 kDa	1.41	1.26	1.18	1.10	1.10	0.71	++	++
30–50 kDa	1.49	1.26	1.26	1.18	1.18	0.94	++	++
10–30 kDa	0.63	0.31	0.24	0.16	0.08	0.00	−	±
<10 kDa	0.08	0.00	0.00	0.00	0.00	0.00	−	±

AF: aquafaba; AF-S: supernatant of aquafaba; AF-P: precipitate of aquafaba; ++ indicates a strongly positive reaction; + indicates a positive reaction; ± indicates a weakly positive reaction; and − indicates a negative reaction.

.

The analysis revealed that the 50–100 kDa and 30–50 kDa fractions exhibited excellent foaming properties. Foam volume was also monitored over 24 h. The 50–100 kDa and 30–50 kDa fractions maintained their foam stability for up to 6 h, with a gradual decrease thereafter, whereas fractions below 30 kDa lost their foam more rapidly. These findings suggest that components contributing to both foaming and foam stability are present in the 30–100 kDa range. In addition, a comparison of the foaming properties between the original aquafaba and the fractions with foaming activity (30–100 kDa) revealed that the 30–100 kDa fractions, particularly the 30–50 kDa fraction, exhibited improved foaming properties compared to aquafaba. This indicates that the concentration of components in the 30–100 kDa range enhances foaming activity. In other words, components below 30 kDa and above 100 kDa do not contribute to the foaming properties, suggesting that the 30–100 kDa fraction represents the primary foaming components in reheated aquafaba.

Qualitative tests were conducted to determine whether the fractions contained proteins and carbohydrates. The results revealed that the 30–50 kDa and 50–100 kDa fractions both exhibited positive colorimetric reactions, whereas no reactions were observed in the other fractions. These findings indicate that the foaming properties of reheated aquafaba are attributed to proteins and carbohydrates in the 30–100 kDa range, which also contribute to foam stability over time.

4. Conclusions

This study comprehensively analyzed the functional properties of aquafaba, focusing on the effects of concentration and enzymatic treatments on its foaming and emulsifying properties, as well as the molecular size of its functional components. The results confirmed that the concentrated components eluted from cooked chickpeas, primarily proteins and sugars, which significantly contribute to enhancing aquafaba’s functional properties. The enzymatic treatment experiments further suggested that aquafaba’s foaming and emulsifying properties are achieved not only by proteins but also through interactions with pectin and other carbohydrates. The degradation of proteins by protease markedly reduced foam formation and emulsion stability, underscoring the essential role of proteins in these processes. Additionally, the molecular size analysis revealed that components with foaming properties are concentrated in the 30–100 kDa range, highlighting the importance of high molecular weight components in the functionality of concentrated aquafaba. These findings illustrate that aquafaba’s functionality arises from complex interactions between its diverse components, with concentration playing a pivotal role in enhancing these characteristics. Although our findings advance our understanding of aquafaba’s functional properties, further detailed investigations are required to fully explore the mechanisms underlying the interactions between proteins and carbohydrates, as well as their synergistic contributions to foaming and emulsifying properties. Such research will provide deeper insights into aquafaba’s potential applications in food systems.