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Article

Valorization of Black Beans (Phaseolus vulgaris L.) for the Extraction of Bioactive Compounds Using Solid-State Fermentation

by
Dulce W. González-Martínez
1,
Alma D. Casas-Rodríguez
1,
Sergio A. Coronado-Contreras
1,
Adriana C. Flores-Gallegos
1,
Claudia M. López-Badillo
1,
Juan A. Ascacio-Valdés
1,
Antonio Flores-Naveda
2 and
Leonardo Sepúlveda
1,*
1
Faculty of Chemical Sciences, Autonomous University of Coahuila, Saltillo, Coahuila 25280, Mexico
2
Center for Training and Development in Seed Technology, Autonomous Agrarian University Antonio Narro, Saltillo 25315, Coahuila, Mexico
*
Author to whom correspondence should be addressed.
Waste 2025, 3(2), 13; https://doi.org/10.3390/waste3020013
Submission received: 2 March 2025 / Revised: 6 April 2025 / Accepted: 9 April 2025 / Published: 11 April 2025
(This article belongs to the Special Issue Agri-Food Wastes and Biomass Valorization—2nd Edition)
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Abstract

:
Black beans (Phaseolus vulgaris L.) are one of the most consumed legumes worldwide. Black beans are rich in proteins, vitamins, minerals, and polyphenolic compounds. The present study aims to valorize black beans for the extraction of polyphenolic compounds using solid-state fermentation (SSF) from Aspergillus niger GH1. A physicochemical analysis of black beans was performed. Fermentation kinetics was performed to establish the best accumulation time of condensed polyphenols. A two-level Plackett–Burman experimental design was used to evaluate the culture conditions (temperature, humidity, inoculum, particle size, pH and salt concentration) for the accumulation of condensed polyphenols. The results of the physicochemical analysis showed that black beans can be used as a substrate in the SSF process. In addition, the best time for the accumulation of condensed polyphenols was 48 h. Treatment 5 achieved an accumulation of 21.04 mg/g of condensed polyphenols. While the factors of particle size, humidity, and temperature had a significant effect on the accumulation of condensed polyphenols. It is concluded that the SSF process is an efficient and eco-friendly extraction method for obtaining bioactive molecules with potential applications in the pharmaceutical, food, and cosmetic industries.

1. Introduction

The production of beans (Phaseolus vulgaris L.) in Mexico in 2024 reached around 1.82 million tons, and beans represent a key crop in the diet of Mexicans [1]. Beans have different varieties; black beans are one of the most important legumes due to their content of proteins, carbohydrates, dietary fiber, minerals, vitamins, and other bioactive compounds [2]. The color of the testa is due to the phytochemical composition and the presence of condensed polyphenols such as anthocyanins and flavonoids, among others [3]. Condensed polyphenols are oligomeric compounds made up of polyhydroxyflavan-3-ol monomeric units linked mainly by C4–C6 or C4–C8 bonds, where their molecular weight ranges from 500 to 20,000 Da [4]. These molecules are important in the pharmaceutical and medical industry due to their biological properties, namely, their anti-inflammatory and antioxidant properties, promoting cardiovascular health and improving gastrointestinal and bone health [5].
To obtain these bioactive molecules, non-conventional methods such as SSF can be used. This methodology is defined as a bioprocess in which microorganisms grow on a solid matrix in a reduced percentage of water [6]. The SSF is used to increase the concentration of bioactive compounds by improving their biosynthesis and/or extractability. This is due to the structural degradation of cell walls caused by fungal growth, the release of bioactive compounds linked to the action of hydrolytic enzymes produced by microorganisms during fermentation, and the production of bioactive compounds synthesized by the microorganism [7]. The SSF process has some advantages, such as increasing productivity, reducing operating costs, reducing water consumption, and using waste as a source of carbon and energy, among others [8].
There are relatively few research works that study the extraction of bioactive compounds from black beans using SSF with filamentous fungi. We can mention some similar works, for example, green kernel black beans were solid fermented with E. cristatum, and the chemical composition and metabolites during fermentation were investigated. The authors mentioned that after fermentation, total polyphenolic and flavonoid contents in green kernel black beans were elevated. E. cristatum fermentation also induced the biotransformation of glycosidic isoflavones into sapogenic isoflavones [9]. On the other hand, the solid-state bioconversion process from black bean seeds using a strain of Rhizopus oligoporus were optimized to obtain a functional flour. The fermentation process increased the total phenolic compounds from 190 to 432 mg EGA/100 g DW [10]. In another research work, the effect of fermented black bean (Phaseolus vulgaris L.) phenolic compounds and protein hydrolysates on markers associated with obesity and T2DM was evaluated. The authors concluded that SSF is a processing method for generating functional ingredients rich in bioactive compounds capable of acting on biological markers related to the treatment or prevention of obesity and T2DM [11].
The main objective of this research work was to valorize black beans as a substrate and to evaluate the conditions of the SSF process using a widely studied filamentous fungus such as Aspergillus niger GH1 for the accumulation of bioactive molecules of polyphenolic origin.

2. Materials and Methods

2.1. Raw Material

The black bean (Phaseolus vulgaris L.) seeds were collected in the 2022 harvest of the Autonomous Agrarian University Antonio Narro and were washed and dried in an oven (Luzeren®, model WGL-65B, Shanghai, China) at 35 °C for 24 h; then, they were ground and stored in airtight jars in refrigeration until later use.

2.2. Proximal and Physicochemical Characterization

At the water absorption index (WAI), 1 g bean samples were taken and placed in 50 mL falcon tubes, and 30 mL of distilled water was added. Samples were manually shaken for 1 min at room temperature and then centrifuged (Premiere®, Model XC-2450, Muskogee, OK, USA) at 5000 rpm for 20 min. The supernatant was decanted, and WAI was calculated considering the gel weight and expressed as grams of gel over grams of dry weight [12]. At the critical humidity point, 1 g of black bean seed, previously ground and dried, was placed on a thermos-balance (OHAUS, Model MB23, Greifensee, Switzerland), in which the weight loss was recorded according to the increase in temperature. For the determination of the protein content, also called organic nitrogen, the Kendhal method was used, according to the methodology of Djao et al. [13]. Subsequently, the lipid and ash content were determined using the methodology described by Fan and Beta [14]. For the determination of total sugars, the phenol–sulfuric method was used according to the methodology of Messou et al. [15].

2.3. Strain Reactivation

The strain Aspergillus niger GH1 from the collection of the DIA UadeC, deposited in the mycological library of the University of Minho with the number MUM: 23.16 was used. The strain on potato dextrose agar (SOLBIOSA) was reactivated. Finally, the strain was placed in an incubator (Yamato Scientific Chongqing Co., Ltd., Chongqing, China) for seven days.

2.4. Kinetics of Accumulation of Condensed Tannins

The tannin concentration kinetics were carried out by placing 3 g of previously ground black beans and mixing it with 7 mL of deionized water in a Petri dish. Subsequently, an inoculum of 1 × 107 spores/g of Aspergillus niger GH1 was placed. The fermentation kinetics were carried out from 0 to 72 h, taking samples every 12 h. The extracts were recovered with 15 mL of 50% ethanol. The concentration of condensed polyphenols was determined using the HCl/Butanol technique, as described in Section 2.6.

2.5. SSF Conditions

The Petri dishes was selected, and 3 g of black bean was placed, which was pre-dried in an oven at 35 °C for 24 h in (Luzeren®, model WGL-65B, Shanghai, China). To carry out SSF, a two-level Plackett–Burman experimental design was carried out with a total of 24 treatments to determine the effect of the culture conditions on the bioprocess for accumulated condensed polyphenols (Table 1). Temperature, humidity, inoculum, particle size, pH, magnesium sulphate, and sodium nitrate were evaluated. The extract was recovered with 50% ethanol by manual pressing after 48 h [16].

2.6. Condensed Polyphenols Content

The condensed tannin content of the extracts was estimated according to the HCl–butanol method with some modifications. A total of 250 µL of the extract was taken, and 1.5 mL of HCl–butanol (1:4) was added; then, the samples were boiled for 60 min, and finally, 190 µL of the sample was taken to subsequently take a reading of the absorbance at 460 nm in a microplate reader (Thermo Scientific MultiSkan, Singapore). Catechin was used for the calibration curve, and the results were expressed as mgCE/g based on dry weight [10]. All results were performed by triplicate.

2.7. HPLC-MS Analysis

The profile of molecules was identified according to the methodology described by Cerda-Cejudo et al. [17]. The samples were filtered with a 0.45 µm membrane. A liquid chromatography equipment (Varian ProStar 3300, Varian, Palo Alto, CA, USA) and a Dynamax Microsorb300 C18 column (250 mm × 21.4 mm, 10 µm) were used. A flow rate of 8 mL/min was used, and the conditions were as follows: as mobile phase, (A) CH3COOH (3% v/v in water), and (B) methanol. The method used for the separation of the molecules was isocratic: 5% initial B; 0–45 min, 5–90% B; 45–50 min, 90% B; 50–70 min, 90–5% B; 70–95 min.

2.8. Statistical Analysis

All treatments in this section were performed in triplicate. A one-way ANOVA test was performed to determine the statistical significance of differences in mean values, and p < 0.05 was considered statistically significant. The interpretation of the results and the construction of the Pareto chart were determined using the statistical package STATISTIC in its version 7.0.

3. Results and Discussion

3.1. Analysis of the Physicochemical and Proximal Characterization

The results of the physicochemical and proximate characterization of black beans are shown in Table 2. The protein content in black beans was 43%; this result may vary according to reports in the literature. In a study of the physicochemical composition of different varieties of beans produced in Mexico, it was found that the protein content of black beans was 39.76% [18]. The lipid and fiber content were 8.14 and 15.57%, respectively. The fiber content in this study was high compared to those of other authors. For example, in a study on the physicochemical characterization of black bean flour, the fiber content was 3.37% [19]. The carbohydrate content was 20.84%; these values are low compared to reports in the literature; for example, some authors mention that the carbohydrate content in black beans can reach values of 74.27% [20]. The ash content was 3.13%; these values are similar to those reported in the literature; for example, in a chemical characterization of black beans, one study found values of 3.3% [21]. Humidity content is a critical factor in the SSF process since proper growth of the filamentous fungus depends on it. In this case, the maximum humidity was 5.4%. These results agree with the values reported by Sánchez-Quezada et al. [22]. The authors evaluated the humidity content in three bean flours with different treatments; the values were in a range of 6.7–8.1%, similar to those found in this study. On the other hand, values of 8.9% humidity have been reported in dehulled hardened beans [23]. Recent studies have evaluated the mineral content in different varieties of black beans. Although specific values may vary depending on the variety and growing conditions, black bean seeds have been found to contain minerals such as calcium, iron, magnesium, and zinc [24]. These differences in mineral concentrations in different bean varieties can be attributed to differences between cultivars/genotypes, the inclusion of different numbers of genotypes, and differences in soil types and environmental conditions [25]. These results demonstrated that black beans are suitable for use as a substrate and source of carbon and energy for the microorganism in the fermentation process.

3.2. Evaluation of Fermentation Conditions for the Accumulation of Condensed Polyphenols

Figure 1 shows the kinetic behavior of the accumulation of condensed polyphenols in the fermentation process with Aspergillus niger GH1. In the first 36 h, a decrease in the content of condensed polyphenols was observed, reaching 7.53 mg/g. At 48 h, a maximum accumulation of 16.09 mg/g was reached. Subsequently, these values fell to 6.74 at 72 h. These fermentation kinetics for the accumulation of condensed polyphenols show that the best time was at 48 h. In a previous work, Aspergillus niger GH1 was able to grow and develop on complex substrates rich in polyphenols from creosote bush (Larrea tridentata Sessé and Moc. ex DC. Coville), pomegranate (Punica granatum L. var Wonderful), and cranberry (Vaccinium macrocarpon Ait.) [26].
Figure 2 shows the accumulation of condensed polyphenols obtained via the Plackett–Burman experimental design. Treatment 5 achieved a condensed polyphenol accumulation of 21.04 mg/g, while treatment 4 only achieved a condensed polyphenol accumulation of 5.31 mg/g. There are very few works in the literature on fermentation processes with Aspergillus niger for the release of condensed polyphenols using black beans as a carbon and energy source. However, we can mention some; for example, some authors have studied the SSF process with filamentous fungi (Aspergillus awamori, A. oryzae, A. sojae, Rhizopus azygosporus, and Rhizopus sp. No. 2) to improve the polyphenol content. The authors concluded that SSF with filamentous fungi improves the levels of bioactive compounds in black beans [27]. The literature mentions that the accumulation of polyphenol content during fermentation is due to enzymes such as β-glucosidase, β-xylanase, and β-arabinofuranosidase, which can be produced by the Aspergillus genus. These enzymes break the bonds between phenolics and their glycosides, increasing the mobilization of bioactive components, releasing phenolics, and improving polyphenol content [28].
In another study, dynamic changes in the sensory properties, nutritional quality, and metabolic profiles of black beans during the SSF process using the fungus Eurotium cristatum were evaluated. One of the main results was the increase in flavonoids from 22.86 to 33.22 mg/g [9]. Furthermore, they evaluated the SSF potential of some legumes, including black beans, using the fungus Cordyceps militaris to increase the content of bioactive compounds. Among the main results, they demonstrated that the total flavonoid content reached a maximum yield of 1.44 mg/g in black beans [29].
Figure 3 shows the Pareto chart of the main estimated effects of the fermentation process on the accumulation of condensed polyphenols. The red dotted line represents the p 0.05 value. All factors evaluated, except humidity, have a significant effect on the accumulation of condensed polyphenols.
Temperature is a crucial factor influencing microbial growth and the production of secondary metabolites and enzymes during SSF. Heat production and difficulties in mass transfer can negatively affect the growth of microorganisms [30]. Nutrients play an important role in the SSF process; they are essential for the growth and metabolic activity of microorganisms. Sodium nitrate is a source of nitrogen for microorganisms, necessary for protein synthesis, while magnesium sulfate is a source of important minerals for sporulation and enzymatic cofactors for various metabolic pathways [6]. Inoculum size is a critical parameter in SSF because it directly influences the start-up and yield of the process. A low inoculum can delay substrate colonization and increases the risk of contamination. Conversely, a high inoculum can lead to mass transfer problems as high cell density limits the diffusion of oxygen and nutrients through the substrate [31]. pH determines the optimal conditions for microorganism growth. Filamentous fungi thrive in slightly acidic conditions, with the optimum range generally being between 3.8 and 6.0 [32]. Finally, the size of the substrate particles directly influences SSF. Smaller particles offer a greater surface area, which favors the colonization and growth of fungi. However, reducing the size also reduces the interparticle spaces, which limits aeration and oxygen diffusion, essential for the respiratory process [33]. Humidity did not have a significant effect on the release of condensed polyphenols. Meini et al. [34] mention that humidity is not an essential variable in the release of polyphenols. Humidity in SSF processes can vary between 30 and 85%.
The analysis of variance (ANOVA) of the regression model is presented in Table 3. The model used in the experiment was as follows: p < 0.05; coefficient of determination R2 = 0.9195; and adjusted coefficient of determination R2Adj = 0.8843. The loss of fit term was not significant, indicating that the model is reliable and can be used to analyze and predict the effect of SSF process conditions on the content of condensed polyphenols using black beans as a substrate. From this analysis, it can be determined that most of the factors had significant effects on the response variable except for humidity. The order of the effect of each factor was temperature > magnesium sulfate > sodium nitrate > amount of inoculum > pH > particle size. Although significant results are obtained, we believe that the yields for the extraction of polyphenolic compounds using SSF can be improved. Some authors mention that optimizing the SSF process is an efficient strategy for improving phenolic content [35].

3.3. Identification of the Main Phenolics Compounds by HPLC-MS

The best fermentation extract (treatment 5) was subjected to identification of the main phenolic compounds by HPLC-MS, as shown in Table 4. Coumarins are not commonly reported compounds in beans. Coumarins are organic compounds belonging to the benzopyrone family, are secondary metabolites of plants, and are widely distributed in nature [36]. Esculin is an organic compound belonging to the coumarin family, specifically a coumarin glucoside. This molecule has different biological properties, such as antioxidant, antimicrobial, anti-inflammatory, and antidepressant properties, among others [37]. The presence of hydroxybenzoic acids in Phaseolus vulgaris is more common and has been reported in the literature. For example, the presence of polyphenolic compounds was evaluated, specifically some hydroxybenzoic acids, such as 4-hydroxybenzoic acid, in different bean varieties [38]. Gallic acid and its derivatives, such as gallic acid 3-O-gallate, have different biological properties, some of which stand out as antioxidant, antimicrobial, and antitumor properties [39]. The presence of ferulic acid and its derivatives has been detected in several varieties of common beans. For example, Yang et al. [40] mention the presence of ferulic acid and its derivatives in several varieties of common bean. Furthermore, ferulic acid derivatives represent a significant percentage of the total phenolic content in raw and cooked beans, highlighting their importance within the phenolic compound profile of this legume.

4. Conclusions

The SSF process with Aspergillus niger GH1 is an efficient and sustainable method for the extraction of bioactive compounds from black beans. This process allowed for the maximum accumulation of condensed polyphenols at 48 h. Treatment 5 reached a maximum of 21.04 mg/g. Furthermore, factors such as temperature, particle size, inoculum, pH, and culture medium composition play a crucial role in the accumulation of condensed polyphenols. HPLC-MS analysis confirmed the presence of esculin, gallic acid 3-O-gallate, and 5, 5′ dehydrodiferulic acid. These findings highlight the potential of SSF as an ecological alternative to valorize black beans and obtain high-added-value molecules. Although fermented black beans have high potential for a variety of applications, it is crucial to ad-dress existing concerns through further research and rigorous testing to confirm their safety before their widespread introduction into food, pharmaceutical, and cosmetic products.

Author Contributions

Conceptualization, D.W.G.-M.; data curation, D.W.G.-M. and L.S.; formal analysis, A.D.C.-R., S.A.C.-C. and A.C.F.-G.; investigation, D.W.G.-M. and L.S.; methodology, D.W.G.-M., C.M.L.-B. and J.A.A.-V.; resources, A.F.-N.; supervision, A.C.F.-G., J.A.A.-V. and A.F.-N.; writing—original draft, D.W.G.-M. and L.S.; writing—review and editing, D.W.G.-M. and L.S. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author(s).

Acknowledgments

D.W.G.M. thanks the SECIHTI of the Mexican government for the scholarship granted to develop his doctorate. The authors thank the UAdeC and the UAAAN for the facilities provided.

Conflicts of Interest

The authors declare no conflicts of interest.

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Waste 03 00013 g001
Figure 1. Kinetics of the accumulation of condensed polyphenols in the fermentation process with Aspergillus niger GH1.
Figure 1. Kinetics of the accumulation of condensed polyphenols in the fermentation process with Aspergillus niger GH1.
Waste 03 00013 g001
Waste 03 00013 g002
Figure 2. Accumulation of condensed polyphenols using a Plackett–Burman experimental design.
Figure 2. Accumulation of condensed polyphenols using a Plackett–Burman experimental design.
Waste 03 00013 g002
Waste 03 00013 g003
Figure 3. Pareto chart of the main estimated effects of the fermentation process on the accumulation of condensed polyphenols.
Figure 3. Pareto chart of the main estimated effects of the fermentation process on the accumulation of condensed polyphenols.
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Table 1. Condensed matrix of treatments and independent factors evaluated using a Plackett–Burman experimental design.
Table 1. Condensed matrix of treatments and independent factors evaluated using a Plackett–Burman experimental design.
TreatmentsTemperature (°C)Humidity (%)Inoculum (Spores/g)Particle Size (mm)pHMgSO4 (g/L)NaNO3 (g/L)
1−1−1−1111−1
21−1−1−1−111
3−11−1−11−11
411−11−1−1−1
5−1−111−1−11
61−11−11−1−1
7−111−1−11−1
81111111
FactorsHigh Level (+1)Low Level (−1)
Temperature (°C)3025
Humidity (%)8060
Inoculum (spores/g)1 × 1081 × 106
Particle size (mm)1.41 mm0.354 mm
pH65
MgSO4 (g/L)15.67.8
NaNO3 (g/L)3.041.50
Table 2. Physicochemical and proximate characterization of black beans.
Table 2. Physicochemical and proximate characterization of black beans.
ParametersResults (%)
Protein43.13 ± 0.88
Lipids8.14 ± 0.38
Fiber15.57 ± 0.04
Carbohydrates20.84 ± 0.87
Ash3.13 ± 0.11
Humidity5.2 ± 0.53
Minerals
Fluorine (F)2.86
Iron (Fe)2.77
Zinc (Zn)1.32
Manganese (Mn)0.78
Copper (Cu)0.44
Strontium (Sr)0.41
Molybdenum (Mo)0.23
Cobalt (Co)0.22
Others 10.80
1 magnesium (Mg); potassium (K); titanium (Ti); nickel (Ni).
Table 3. Analysis of variance (ANOVA) of the regression model.
Table 3. Analysis of variance (ANOVA) of the regression model.
FactorsSSdFMSFp
(1) Temperature (°C)217.74581217.745872.06100.000000
(2) Humidity (%)3.191613.19161.05620.319364
(3) Inoculum (spores/g)65.7839165.783921.77060.000258
(4) Particle Size (mm)17.6898117.68985.85420.027814
(5) pH59.0046159.004619.52700.000430
(6) MgSO4 (g/L)122.86301122.863040.66040.000009
(7) NaNO3 (g/L)65.8138165.813821.78050.000258
Error48.3469163.0217
Total SS600.439323
Table 4. Tentative identification of the main phenolic compounds by HPLC-MS.
Table 4. Tentative identification of the main phenolic compounds by HPLC-MS.
Tentative IdentificationStructure 1Retention TimeElemental Formula[M-H]−
EsculinWaste 03 00013 i0015.38C15H16O9338.9
Gallic acid 3-O-gallateWaste 03 00013 i00217.66C14H9O9320.1
5-5′Dehydrodiferulic acidWaste 03 00013 i00321.8C20H18O8385.1
1 Structures obtained in PubChem [41]. (https://pubchem.ncbi.nlm.nih.gov).
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González-Martínez, D.W.; Casas-Rodríguez, A.D.; Coronado-Contreras, S.A.; Flores-Gallegos, A.C.; López-Badillo, C.M.; Ascacio-Valdés, J.A.; Flores-Naveda, A.; Sepúlveda, L. Valorization of Black Beans (Phaseolus vulgaris L.) for the Extraction of Bioactive Compounds Using Solid-State Fermentation. Waste 2025, 3, 13. https://doi.org/10.3390/waste3020013

AMA Style

González-Martínez DW, Casas-Rodríguez AD, Coronado-Contreras SA, Flores-Gallegos AC, López-Badillo CM, Ascacio-Valdés JA, Flores-Naveda A, Sepúlveda L. Valorization of Black Beans (Phaseolus vulgaris L.) for the Extraction of Bioactive Compounds Using Solid-State Fermentation. Waste. 2025; 3(2):13. https://doi.org/10.3390/waste3020013

Chicago/Turabian Style

González-Martínez, Dulce W., Alma D. Casas-Rodríguez, Sergio A. Coronado-Contreras, Adriana C. Flores-Gallegos, Claudia M. López-Badillo, Juan A. Ascacio-Valdés, Antonio Flores-Naveda, and Leonardo Sepúlveda. 2025. "Valorization of Black Beans (Phaseolus vulgaris L.) for the Extraction of Bioactive Compounds Using Solid-State Fermentation" Waste 3, no. 2: 13. https://doi.org/10.3390/waste3020013

APA Style

González-Martínez, D. W., Casas-Rodríguez, A. D., Coronado-Contreras, S. A., Flores-Gallegos, A. C., López-Badillo, C. M., Ascacio-Valdés, J. A., Flores-Naveda, A., & Sepúlveda, L. (2025). Valorization of Black Beans (Phaseolus vulgaris L.) for the Extraction of Bioactive Compounds Using Solid-State Fermentation. Waste, 3(2), 13. https://doi.org/10.3390/waste3020013

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