SJA_39_s1_208-219

Introduction

Cowpea [(Vigna unguiculata) (L.) Walp.] with common name: black-eye pea, southern pea, niebe, and crowder pea (in Indonesian: kacang tunggak, kacang tolo, and kacang merah kecil) is a leguminoceae crop that is strongly potential to be a protein source (Mfeka et al., 2019). It has yet not been commercially developed by farmers despite being cultivated in Indonesia for quite some time. With protein content varied between 22.37 % and 24.54 % (Gerrano et al., 2022), cowpea planted in Brazil holds 20.82 % to 26.92 % (Freitas et al., 2022) while in Indonesia is of 22.90 % in addition to 11 % water, 1.4 %

fat, and 61.60 % carbohydrate (Kanetro, 2017). Its lower fat content compared to other legumes should reduce the risk commonly occur after consuming fatty foods. Moreover, cowpea contains more vitamin B1 than mung bean [Vigna radiata (L.) R. Wilczek] while its protein is rich in essential amino acids , such as lysin, aspartat acid, and glutamat (Rosida et al., 2013; Setyobudi et al. 2023, 2024a).

Food processing is no longer limited to conventional methods technology currently allows legumes to be stored in the form of food ingredients, e.g. flour, concentrate, or protein isolate (Burger et al., 2022; Damat et al., 2023; Strauch and Lila, 2021; Witono et al., 2014). Protein isolate is an isolation product of legumes generally employing extraction (alkaline-based) and coagulation (acid-based) methods with a standard protein content of 90 %. Most protein isolate in Indonesian market is imported due to inadequate local supply (Bahar and Witono, 2015). After undergoing an acetone wash and 8 h drying at 50 °C, cowpea contains 16.638 % protein isolate yield as well as 7.932 % water, 90.305 %

protein, 1.050 % fat, 8.784 % carbohydrate, and 3.852 % ash (Anam et al., 2013). To compare, a green pea (Lathyrus oleraceus Lam.) protein isolate product in Chicago, USA is of 81.3 % protein, 7 % fat, and 9 %

fiber (Masia et al., 2022).

Further on cowpea protein isolate, it is also noted to contain more 7S globulin fraction than 11S one. However, its gelatinization ability is as low as 4 gf. Also recorded technical functional characteristics are pH saturation (pH 8), foam capacity (68 mL g–1), foam stability (8 %), oil holding capacity (84.89 %), water holding capacity (136.61 %), emulsion capacity (2.41 m2 g–1) and emulsion stability (78.15 h) (Witono et al., 2014).

Chemical modification on functional features (Wu et al., 2023) with CH3COOH, CaSO4 (Chang et al., 2011; Hou et al., 1997), and MgSO4 is a way to boost gelatinization ability of protein (Arabi et al., 2020; Bangar et al., 2022; Hsieh et al., 2022). Acetic acid (CH3COOH) reduces the negative charge to enhance gelatinization, calcium sulfate (CaSO4) cross-links Ca+ ion and carboxyl group to aggregate and form gel. Adding magnesium sulfate (MgSO4) has the same effect as the latter. Concentrations of 3 %

to 7 % for acetic acid and 0.25 % to 0.35 % for both calcium sulfate and magnesium sulfate allow a wide range of modification results to explore leading to an optimal protein modification process.

The best modification on cowpea is of 0.35 % CaSO4 and 0.35 % MgSO4 combination, resulting in an isolate of 13.9 gf mm-1 gel strength containing 81.04 %

water, 6.77 % ash (db), 92.53 % protein (db), and soluble protein in pH 8 at 22.47 mg g–1 (db) (Anam et al., 2024). Since the functional features of the aforementioned treatment are unknown, this study aims to reveal them in modified cowpea protein isolate in accordance with certain chemical types and concentrations. This research is urgently needed to support the development of healthy local plant based protein ingredients, enhance the competitiveness of indigenous legumes, and contribute to reducing dependency on imported protein isolates in the Indonesian food industry. The broader goal is to achieve Sustainable Development Goals, especially the 2nd Goal: Zero hunger; the Goal 3: Good health and well being; the Goal 9: industry, innovation, and infrastructure; and the Goal 12: Responsible consumption and production (United Nations, 2025).

Materials and Methods

The exploration on varied chemical types and concentrations in modifying cowpea protein isolate had been performed between May and August 2022 in the Food Processing and Agricultural Product Engineering Laboratory, the Food Chemistry and Biochemistry Laboratory, and the Integrated Analysis Laboratory of the Faculty of Agriculture of Universitas Jember, focusing on foam capacity, foam stability, oil holding capacity, water holding capacity, emulsion capacity and emulsion stability.

Materials

Cowpea sample was locally obtained from Malang, East Java, Indonesia. Chemicals involved were aquadest, aceton 70 %, NaOH 0.1 N, HCL 1 N, H2SO4, phosphate buffer 0.1 M pH 7, phosphate buffer 0.05 M pH 7, CH3COOH, CaSO4, and MgSO4 all of pro analiysis quality purchased from PT. Pash Mitra Mandiri, Bandung, West Java. Equipments used were blender (GMC, China), filtering cloth, pH meter (Jen Way 3320, Germany), centrifuge (Medifringer Gyrozen 2236 HR, South Korea), centrifuge tube (China), spatula, water bath (Memmert, Jerman), stirrer (SM 24, Germany), stirrer shaft (SM 24, Germany), analytic scale (Ohaus Ap-310-O, AS), vortex (Eppendorf, Germany), spectrophotometer (Genesis 10 UV, USA), and glassware.

Methods

This study employed a Completely Randomized Design (CRD) with a single factor: the type and combination of chemical modifiers. The experiment consisted of three treatments, each replicated three times, resulting in a total of nine experimental units. The treatments were as follows: Treatment A = 0.35 % calcium sulfate (CaSO₄) + 7 % acetic acid (CH₃COOH); Treatment B = 0.35 % magnesium sulfate (MgSO₄) + 7 % acetic acid (CH₃COOH); and Treatment C = 0.35 % calcium sulfate (CaSO₄) + 0.35 % magnesium sulfate (MgSO₄). Each experimental unit consisted of 100 g of dried cowpea seeds, which were processed into protein isolate and then chemically modified according to the designated treatment. The variables in this study were: i) Independent variable: The type and concentration of chemical reagents (CaSO₄, MgSO₄, CH₃COOH); and ii) Dependent variables: Foaming capacity, foaming stability, oil holding capacity (OHC), water holding capacity (WHC), emulsion capacity, and emulsion stability The experimental procedure was carried out in three main stages: i) Protein Isolation: Cowpea protein isolate was extracted using an alkaline extraction and acid precipitation method, following Witono et al. (2014) and Shanthakumar et al. (2022); ii) Chemical Modification: The extracted protein isolate was modified using specific combinations and concentrations of CaSO₄, MgSO₄, and CH₃COOH according to the treatment groups; and iii) Functional Property Analysis: The modified protein isolate was evaluated for its functional properties, including foaming capacity and stability, OHC, WHC, emulsion capacity, and emulsion stability. Measurements and calculations were performed using standardized analytical methods based on relevant scientific literature.

Sample preparation (Witono et al., 2014)

An sample of 100 g cowpea was soaked in water for ± 5 h. Once drained, the rind was extracted at the ratio of 3:1 (w w–1) and filtered. The supernatan was mixed with NaOH 0.1 N at the ratio of 0.5:1 in a glass beaker to optimally solve the protein in pH 8, then heated at 55 oC for 30 min. Centrifugation followed at 2 000 rpm (1 Hz = 60 rpm) for 10 min. HCl 1 N was added to bring protein precipitation to the isoelectric point of pH 5 before the second centrifugation at 2 000 rpm for 10 min. After purified using acetone 70 % at the ratio of 3:1, homogenously stirred for 20 min , and centrifugated at 2 000 rpm for another 10 min, cowpea protein isolate was ready (Shanthakumar et al., 2022).

Step 1: Sample grouping modification (Anam et al., 2024)

In treatment A, CaSO4 0.35 % and CH3COOH 7 %

were combined. Treatment B received MgSO4 0.35 % and CH3COOH 7 %, while MgSO4 0.35 % and CaSO4 0.35 % were administered in Treatment C. After homogenously mixed using a spatula, each treatment was heated at 70 °C for 15 min to denaturate protein, allowing molecular interaction to form modified cowpea protein isolate gel.

Step 2: Functional feature analysis

Foam capacity and foam stability (Damat et al., 2000; Zayas, 1997)

The amount of 0.5 g sample and 100 mL phosphate buffer 0.1 M pH 7 were mixed in a 250 mL measuring cup (scaled prior to the process) and stirred for 10 min.

Aerator worked in the mixture for 60 s to form foam, of which height was recorded. Both stirrer and aerator were then stopped for 2 min and the foam height reduction was also recorded. The figures were then calculated based on Equation (1) and Equation (2)

...(1)

..........(2)

Emulsion capacity and emulsion stability (Anam et al., 2023a; Parkington et al., 2000)

An amount of 0.2 g sample dan 100 mL phosphate buffer 0.05 M pH 7 were mixed for 15 min, and then blended with 25 mL cooking oil for 3 min. Next, 1 mL

of it was procured for emulsion capacity test, while another 1 mL was taken after resting for 10 min for emulsion stability test. 5 mL Sodium Dodecyl Sulfate SDS 0.1 % was added in each solution prior to vortex round, then absorbance test was performed at 500 nm.

The results were processed as per Equation (3) and Equation (4):

...........(3)

.......(4)

Notes:

c = protein concentration (g mL–1)
φ = oil volume fraction (mL/mL) of emulsion
abs = absorbance
dilution = solution fraction (SDS + emulsion)
EAI = Emulsifying Activity Index
ESI= Emulsifying Stabilitation Index

Oil holding capacity (Anam et al., 2025; Zayas, 1997)

In a prescaled clean centrifugal tube (a g), 2 g sample (b g) and 14 g cooking oil (7× of sample volume) were vortex-mixed and centrifuged for 5 min s at 2 000 rpm. The precipitation was then measured after separated from its supernatant (c g) and run in Equation (5) (Anam et al., 2023b):

.........(5)

Water holding capacity (Ma et al., 2022)

As must as 2 g sample (bg) and 14 g aquadest (7× of sample volume) were vortex-mixed in a prescaled clean centrifugal tube (a g), and centrifuged for 5 min at 2 000 rpm. The precipitation was then measured after separated from its supernatant (c g) and evaluated using Equation (6):

.........(6)

Data analysis

Laboratory experimental research design was utilized, and the data were computed on Microsoft Excel to be presented in the form of tables and graphs. ANOVA test One Way (DSAASTAT 1.101 version, Italy) and Duncan test (p ≤ 0.05) ensured any significance should different results appeared (Damat et al., 2021).

Results and Discussion

Preliminary test (stage 1 and stage 2)

In the first stage, the optimal treatment for cowpea protein isolate was determined based on the firmness and gel strength resulting from the application of different coagulating agents. The objective was to identify which concentration and type of modifier could produce the most compact, rigid, and stable gel structure, as these characteristics are essential indicators of protein denaturation effectiveness and network formation, which directly affect functional properties. Among the tested acid concentrations (3 %,

5 %, and 7 % CH₃COOH), the 7 % CH₃COOH treatment was found to be the most effective. It produced gels with the highest viscosity and firmness, indicating optimal protein precipitation at pH levels conducive to isoelectric point aggregation. This level of acidity effectively disrupted protein solubility and promoted dense gel network formation. For the salt-based coagulants, both CaSO₄ and MgSO₄ were tested at 0.25 %, 0.30 %, and 0.35 %. The 0.35 %

concentration of both salts yielded the strongest and most cohesive gel structures, suggesting that this concentration is most effective in inducing protein cross-linking through ionic interaction, which enhances the protein matrix’s integrity. At this concentration, the divalent cations (Ca²+ and Mg²+) facilitated stronger electrostatic interactions between protein molecules, leading to better gel compactness. Thus, CH₃COOH at 7 % and CaSO₄ and MgSO₄ at 0.35 % were considered effective in enhancing the gelation capacity, structural integrity, and compactness of cowpea protein isolate. These properties are critical for ensuring good functional behavior in food.

The second stage revealed treatment C of CaSO4 0.35 % and MgSO4 0.35 % combination to be the best modification. The gel structure of 13.9 gf. (0.1 mm)–1

is the most compact and, therefore, the most beneficial when applied in the food industry. Having the lowest water content at 81.04 %, this isolate should be the most stable with the longest shelf-life. The highest ash content at 6.77 % (db) points to a significant amount of essential minerals. Its highest protein content at 92.53 % (db) is undoubtedly a feasible protein concentration, with soluble protein content in pH 8 at 22.47 m g–1 (dB), showing its solubility in specific pH environments critical to several food production applications (Anam et al., 2024).

Functional property analysis

Foaming capacity and foaming stability

Two key functional properties of protein in aerated food systems are foaming capacity, which reflects the ability of a protein to entrap air during whipping, and foaming stability, which refers to the protein’s ability to maintain the foam structure over time (Stone et al., 2015). In this study, the foaming capacity of modified cowpea protein isolate ranged from 187 mg g-1 to 227 mg g-1, with a statistically significant difference among treatments (P < 0.05), as shown in Figure 1.

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination
Numbers followed by the same letter indicate no significant difference with the Duncan 5 % test.

Treatment C (0.35 % CaSO₄ + 0.35 % MgSO₄) demonstrated the highest foaming capacity at

227 mg g-1, followed by Treatment B (0.35 % MgSO₄ + 7 % CH₃COOH) and Treatment A (0.35 % CaSO₄ + 7 % CH₃COOH). The enhanced foaming performance in Treatment C may be attributed to the synergistic cross-linking effect of Ca²+ and Mg²+ions, which bind to negatively charged carboxyl groups in proteins, leading to more compact protein networks capable of stabilizing air bubbles (Arabi et al., 2020). These findings are consistent with previous research: The foaming capacity of unmodified cowpea isolate was reported at only 68 mL g-1 (Witono et al., 2014), While Soy (Glycine max Merr.) protein isolate reached 136 mL g-1, and Lima bean (Phaseolus lunatus L.) protein isolate was recorded at 425 mg g-1 (Ajayi et al., 2025), further supporting the functional enhancement through chemical modification.

Regarding foaming stability, the modified cowpea protein isolates showed a range from 20 % to 30 %

(Figure 2), although no statistically significant differences were observed among treatments (P > 0.05).

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination

Treatments B and C exhibited the highest foam stability (30 %), while Treatment A displayed the lowest (20 %). The differences in stability may be due to variations in protein solubility and globulin content, which affect the interfacial film strength (Chao et al., 2018). These results align with Nazar and Usha (2018), who reported that legume protein isolates enriched with 7S and 11S globulins exhibit better foam stability due to their surface-active properties.

In terms of application: Treatments B and C with higher foam stability are suitable for aerated food products requiring stable foam structures, such as mousses, whipped toppings, ice cream, and sausage emulsions, Whereas Treatment A, with quicker foam collapse, is more appropriate for bakery applications such as cakes, where controlled defoaming during baking is desirable.

The improved foaming capacity and stability of cowpea protein isolate particularly in Treatment C suggest its promising potential as a local, plant-based alternative to imported protein isolates in functional food development. However, this study is limited by the absence of evaluation on: Digestibility, Sensory acceptability, and Long-term shelf-life of the modified isolates. Future studies should investigate these parameters to fully assess the commercial applicability of modified cowpea protein isolates in food systems.

Emulsion capacity and emulsion stability

Chemically modified cowpea protein isolates exhibited significantly different emulsion capacities (P < 0.05), ranging from 2870 m² kg-¹ to 5400 m² kg-¹ (Figure 3). Treatment A (CH₃COOH + CaSO₄) showed the highest emulsion capacity (5400 m² kg-¹), significantly outperforming Treatments B and C. This result suggests that acetic acid and calcium sulfate effectively improve interfacial properties of proteins for emulsion formation.

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination
Numbers followed by the same letter indicate no significant difference with the Duncan 5 % test.

The superior performance of Treatment A supports Witono et al. (2014), who found that protein complexity enhances emulsification, and aligns with D’Alessio et al. (2022), who demonstrated that proteins with greater solubility and surface activity form finer emulsions. This finding is also consistent with the emulsion capacity of soybean protein isolates (4 500 m² kg-¹ to 5 600 m² kg-¹) reported by Fu et al. (2020), indicating that modified cowpea protein can match commercial plant protein performance.

Emulsion stability results ranged from 67.20 h to 77.41 h (Figure 4), with Treatment A again producing the most stable emulsion. The presence of globulins 7S and 11S, known to improve interfacial film formation and retard coalescence, likely contributed to this stability (Chao et al., 2018; Witono et al., 2014). Park et al. (2025) also emphasized that legume proteins with higher globulin content enhance emulsion durability.

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination.
Numbers followed by the same letter indicate no significant difference with the Duncan 5 % test.

Despite promising results, the study has limitations. Measurements were taken under controlled conditions, which may not reflect real food systems with variable pH, salt, or fat contents. Microstructure and droplet size distribution were not assessed, which are key parameters in determining long-term emulsion behavior. Future studies should examine stability in actual food matrices, explore sensory implications, and evaluate encapsulation potential for functional food development. Scaling up and shelf-life assessments in commercial formulations are also warranted.

Oil holding capacity (OHC)

Oil holding capacity (OHC) indicates a protein’s ability to retain oil within a food matrix, which is vital for texture and flavor retention in high-fat food systems. In this study, OHC values of chemically modified cowpea protein isolates ranged from 82.16 %

to 92.46 %, with significant differences among treatments (P < 0.05), as shown in Figure 5.

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination.
Numbers followed by the same letter indicate no significant difference with the Duncan 5 % test.

Treatment C (combination of 0.35 % CaSO₄ and 0.35 % MgSO₄) showed the highest OHC value of 92.46 %, significantly greater than Treatment A (CH₃COOH + CaSO₄) and Treatment B (CH₃COOH + MgSO₄). This superior performance may be attributed to the synergistic effect of divalent salts, which promotes the formation of a denser and more uniform protein gel network, increasing surface area for oil entrapment. According to Arabi et al. (2020), smaller protein particle size enhances the interaction with oil due to increased surface reactivity. These findings align with Wisaniyasa and Suter (2016), who reported that common bean sprout powder had an OHC of 89.84 %, and regular bean powder had 86.03 %. Furthermore, the OHC of modified cowpea protein in this study surpassed that of soy protein isolate (84.89 %) as previously reported by Witono et al. (2014), indicating its potential use in high-fat plant-based products such as vegan sausages, spreads, and bakery fillings. This highlights the role of salt-induced protein gelation in enhancing fat-binding capacity, a crucial feature for texture control in formulated foods (D’Alessio et al., 2022).

Future studies should investigate: i) The correlation between protein microstructure (e.g., via SEM) and OHC, ii) Performance in real food systems under industrial conditions, iii) Digestibility and sensory acceptance of oil rich formulations using this modified isolate. Limitation: This study did not include microstructural characterization of the modified proteins, which would have helped explain the observed oil retention mechanism at the molecular and physical levels.

Water holding capacity (WHC)

Water holding capacity reflects a protein’s ability to retain moisture within a food matrix, a critical attribute in formulating plant based foods. In this study, WHC values of the chemically modified cowpea protein isolate ranged from 5.82 % to 60.67 %,

showing significant differences among treatments (P < 0.05) as shown in Figure 6.

Note:

A = CH3COOH - CaSO4 combination; B = CH3COOH - MgSO4 combination; C = CaSO4 -MgSO4 combination
Numbers followed by the same letter indicate no significant difference with the Duncan 5 % test.

Treatment C (0.35 % CaSO₄ + 0.35 % MgSO₄) yielded the highest WHC at 60.67 %, significantly higher than Treatment B (CH₃COOH + MgSO₄), though not significantly different from Treatment A (CH₃COOH + CaSO₄). This indicates that salt-based coagulants (CaSO₄ and MgSO₄) are more effective in enhancing water retention than acid-based coagulants. Both calcium and magnesium ions promote protein cross linking and structure stabilization, allowing proteins to trap more water molecules during gel formation. According to Witono et al. (2014), higher protein content and more compact gel networks result in better water retention. The trend is consistent with Gao et al. (2021), who reported that increasing salt concentration improved WHC in silver carp (Hypophthalmichthys molitrix Valenciennes, 1844) surimi from 85.02 % to 93.63 %. Likewise, Sosa et al. (2020) demonstrated that modified legume proteins could retain water efficiently, supporting their application in moisture-sensitive products like nuggets and meat substitutes.

However, this study did not include microstructural analysis (e.g., via SEM) or test WHC in real food systems. Thus, further research is recommended to: i) Correlate protein gel microstructure with WHC behavior, ii) Evaluate WHC in formulated foods like sauces or protein drinks, and iii) Assess WHC under processing conditions such as heating and storage.

To achieve the goal of producing healthy flour related to SDGs, a Standard Operating Procedure (SOP) must be established, especially to minimize new ubiquitoes pollutants (Chen, et al., 2022; Setyobudi, et al., 2024b, 2024c), i.e., microplastics (MPs). This pollutant has been reported to contaminate soybeans and their derivatives (Setyobudi et al., 2024d). Several researchers, including Hermayanti et al. (2024) and Garfansa et al. (2024), have reported deaths in experimental animals due to MPs contamination.

Conclusions and Recommendations

The optimal chemical modification for cowpea protein isolate was found in Treatment C, engaging 0.35 % CaSO4 and 0.35 % MgSO4 combination. The tested functional properties are of foaming capacity rate at 227 mL g–1 (wb), foaming stability rate at

30 % (wb), oil holding capacity rate at 92.46 % (wb), water holding capacity at 60.67 % (wb), emulsion capacity rate at 2.87 m2 g-1 (wb), and emulsion stability rate at 67.20 h (wb). It is recommended that modified cowpea protein isolate (especially Treatment C) be utilized by the food industry as an alternative to imported protein. Further research is needed on digestibility, sensory characteristics, and industrial feasibility. Government policy support is essential to promote innovation, production, and the use of local legumes as part of a national strategy for protein self-sufficiency and sustainable food industry development.

Acknowledgments

This research was supported by funding from the Universitas Islam Darul ‘Ulum, Lamongan, Indonesia (0234/U/K.1/XII/2021/SK). Thank you to RP Editage for preparing and guiding the scientific writing of this manuscript.

Novelty Statement

This research develops cowpea protein isolate as a functional food ingredient through chemical modification with an optimal combination of CaSO₄ and MgSO₄ (0.35 %), which successfully increases the foam capacity, emulsion, and oil and water absorption. This finding opens up opportunities for using cowpeas in high value added food products while supporting the sustainability of agriculture in dry areas and increasing the economic value of cowpeas.

Authors’ Contribution

Choirul Anam: Designed the study, performed a literature search, wrote the manuscript draft and manuscript revised.

Yuli Witono: Supervised the experiment, analyzed and interpreted the data, and provided comments and suggestions for the whole work.

W.ahyu Mushollaeni, Miftha Churochman, and Asma Nisar: Manuscript review.

Emmy Hamidah, Mariyatul Qibtiyah, and Dian Eka Kusumawati: Conducted the experiments and collected the data.

Istiqomah Istiqomah and Sayekti Nugrahaning Widi: Resources, Grammarly, and Turnitin.

Trias Agung Pakarti: Formatted manuscript and edited in English.

All authors have read and approved the final manuscript

Generative AI and AI-assisted technology statement

The authors stated that they didn’t use generative AI and AI-assisted technology in preparing this manuscript.

Conflict of interest

The authors have declared no conflict of interest.

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