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Solid-State Fermentation as a Sustainable Tool for Extracting Phenolic Compounds from Cascalote Pods

Laboratorio de Biotecnología y Biología Molecular, Departamento de Ciencias Básicas, Universidad Autónoma Agraria Antonio Narro, Saltillo 25315, Mexico
Laboratory of Biotechnology & Bioengineering, Research Center in Food and Development A.C., Delicias 33089, Mexico
Bioprocess & Bioproducts Research Group, Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280, Mexico
Authors to whom correspondence should be addressed.
Fermentation 2023, 9(9), 823;
Submission received: 26 July 2023 / Revised: 4 September 2023 / Accepted: 5 September 2023 / Published: 8 September 2023
(This article belongs to the Special Issue New Trends in Solid Fermentation)


Cascalote pods are an important source of phenolic compounds, mainly recovered using solvent extraction methods. To find a sustainable alternative to these methods, this study aimed to evaluate solid-state fermentation (SSF) in order to enhance the extractability of total phenolic compounds (TPC) with antioxidant activity (AA) from cascalote pods. Aspergillus niger GH1 was selected based on the amount of TPC released and AA measured (ABTS, DPPH, FRAP) in a short period of time. Moreover, moisture, temperature, inoculum size, and mineral composition were evaluated. The largest amount of TPC released was 124.17 mg/gdw (g of dry weight) after 12 h of culture, which significantly correlated with the AA (Pearson’s R = 0.94). Moisture and KH2PO4 concentration were the main influencing factors of TPC release. Treatment 6 (1 × 107 spores/gdw, 30 °C, 60% moisture, mineral composition (g/L): KH2PO4, 1.52; NaNO3, 7.65; and MgSO4, 1.52) was selected due to the highest values of both TPC and AA. SSF-assisted extraction allowed for an increase of 118% and 93% in TPC and AA values, respectively. Corilagin, lagerstannin, geraniin, and ellagic acid were the main phenolic compounds identified by RP-HPLC-ESI-MS in the cascalote extracts. The results obtained demonstrate the feasibility of SSF-assisted extraction as a biotechnological alternative for the recovery of important bioactive molecules from this underutilized material.

1. Introduction

Caesalpinia coriaria is a plant found in the Pacific coastal plains of Central America, commonly called “cascalote” or “divi-divi”, and it is used to obtain firewood, charcoal, poles, and beams. This plant produces pods 3–7 cm long, with a green color when unripe and dark brown when ripe. According to Palma García [1], one cascalote tree can produce up to 150 kg of pods, which indicates a large production.
These pods are commonly used in leather tanning and fodder and are traditionally used as a treatment for infectious skin problems [2]; however, most of the pods produced are underutilized and, consequently, not fully exploited. Additionally, economic activity derived from harvesting has declined. Cascalote pods are astringent due to their high content of phenolic compounds—important molecules that are of great interest in health studies, mainly due to their biological activities such as antimicrobial, antioxidant, and anti-inflammatory [3,4]. The main phenolic compounds that have been reported in cascalote fractions with biological activity are gallic acid, ethyl gallate, stigmasterol, tannic acid, and corilagin [2,5].
Phenolic compounds are commonly recovered using solvents and several extraction techniques, such as maceration, microwave, and ultrasound, among others; however, their use involves expensive equipment as well as environmental pollution and toxicological safety concerns regarding the use of solvents [6,7]. As a biotechnological alternative, the use of fermentative or enzymatic methods for the assisted extraction of biomolecules from vegetal matrices has been reported, which includes solid-state fermentation (SSF) [8].
SSF does not require the use of solvents and promotes high yields and the easy recovery of bioactive compounds. During the SSF process, polyphenols could be biosynthesized or biotransformed into simpler molecules using the activity of microbial enzymes [9,10], which degrade the wall cell components, thus increasing the extraction of free and bound polyphenols [11]. Recently, SSF has been successfully used to obtain bioactive compounds from rambutan peel [12], orange peel [13], pomegranate husk [14], and pineapple waste [15], among others; however, the use of SSF in cascalote pods as a substrate to obtain bioactive compounds has not been studied. Based on responsible production and consumption, this work aimed to develop an eco-friendly bioprocess, SSF, for the extraction, recovery, and identification of compounds with antioxidant capacity from cascalote pods.

2. Materials and Methods

2.1. Raw Material

Cascalote pods were collected in San Miguel Totolapan, Guerrero, México (18.152094485006664, −100.36604425470092), and transported to the laboratory of Biotechnology and Bioengineering in Delicias, Chihuahua, Mexico. They were dried at 50 °C for 24 h (Shel-Lab 1380FX Forced-Air Oven, Cornelius, OR, USA), pulverized (PULVEX Mini 100, Mexico City, Mexico) to a particle size of 1 mm, and stored in a hermetic black polyethylene bottle at room temperature (30 °C) until use.

2.2. Physicochemical Characterization of Cascalote Pods

Protein, fat, carbohydrate, fiber, moisture, and ash content were determined according to the procedures established by the Association of Official Agricultural Chemists (AOAC) [16]. The critical humidity point (CHP) and water absorption capacity (WAC) were obtained according to Orzua et al. [17]. The maximum material moisture was obtained using solids, moisture content, and WAC values [12].

2.3. Microorganisms

For this study, the following strains were used: Aspergillus niger GH1, Aspergillus awamori, and Aspergillus niger HT4 (belonging to the DIA-UAdeC collection). The GH1 strain of A. niger was deposited in the Micoteca of the University of Minho with the number MUM:23.16. Fungal strains were cryopreserved at −55 °C in a skim milk/glycerol (9:1 v/v) solution. Fungal spores were activated on potato dextrose agar (PDA-Bioxon™) plates at 30 °C for 5 days. For the inoculum preparation, fungal spores were collected with a sterile solution of Tween-80 (0.01% v/v) and counted in a Neubauer chamber.

2.4. Solid-State Fermentation (SSF)

Cascalote pods mixed with vermiculite (50:50 w/w) were used as support and as the sole carbon source for fungal growth. SSF was performed in column reactors (31.5 × 180 mm) packed aseptically with a homogeneous mixture containing 6 g of fermentable mass with an initial moisture of 50% (3 g of support impregnated with 3 mL of Czapek–Dox saline solution, previously inoculated with 1 × 106 spores/g of support). A saline solution was prepared to contain (g/L): KCl (1.51), MgSO4 (1.52), NaNO3 (7.65), and K2HPO4 (3.04). SSF was monitored at 30 °C for 72 h, withdrawing samples every 12 h to determine the total phenolic content and antioxidant activity. To obtain the fermented extracts, 12 mL of 50% ethanol was added to each reactor. Samples were shaken in a vortex and filtered through cotton and filter paper (0.45 μm) before being poured into 2 mL vials and stored at −18 °C until analysis.

2.5. Evaluation of the SSF Factors for the Total Polyphenol Extraction

Once the fungal strain had been selected, the time of the maximum release of phenolic compounds and the antioxidant activity were selected as well. Other SSFs were conducted in parallel to identify the effects of inoculum, temperature, and moisture, and the concentrations of KCl, MgSO4, NaNO3, and K2HPO4, according to Table 1. The extracts were obtained after 12 h of SSF with A. niger GH1 and used to determine the TPC and the activity in antioxidant assays.

2.6. Analytical Analysis

2.6.1. Determination of Polyphenol Content

The hydrolysable polyphenols (HP) assay was carried out according to Wong-Paz et al. [18]. In a 96-well microplate, 20 μL of the sample, 20 μL of the Folin–Ciocalteu reagent (2 N), 20 μL of a 20% sodium carbonate solution, and 125 μL of distilled water were mixed. After 5 min of incubation, the absorbance was measured at 790 nm in a microplate reader (Multiskan GO, Thermo Scientific, Waltham, MA, USA). The results were expressed as mg of gallic acid equivalents/g of dry weight (mgGAE/gdw) using a calibration curve (0.04–0.20 mg/mL; R2 = 0.99).
The condensed polyphenols (CP) were determined using a ferric reagent and HCl-butanol (1:9 v/v) [12]. The sample (250 μL) was mixed with 1500 μL of HCl-butanol, and 50 μL of the ferric reagent in 20% HCl was added. The mixture was sealed hermetically and boiled for 40 min. Finally, the samples were cooled, and 200 μL was placed in a 96-well microplate; the absorbance was recorded at 460 nm using a microplate reader (Multiskan GO, Thermo Scientific). The results were expressed as mg of catechin equivalents/g of dry matter (mgCE/gdw) using a calibration curve (0.2–1 mg/mL; R2 = 0.99). The TPC was determined by adding the values of the hydrolysable and condensed polyphenols and expressed as mg/g of dry weight (mg/gdw).

2.6.2. Antioxidant Activity

The antioxidant activity of the extracts was evaluated based on DPPH, ABTS, and FRAP assays. For the DPPH assay, 60 µM DPPH (2,2-diphenyl-1-picrylhydracyl (Sigma-Aldrich®, Naucalpan de Juarez, México)) stock solution (2.365 mg dissolved in 100 mL absolute ethanol) was prepared. The sample (7 μL) and DPPH stock solution (193 μL) were placed in a 96-well microplate, mixed, and kept in darkness for 30 min at room temperature [19]. A microplate reader (Multiskan GO, Thermo Scientific) was used to measure the absorbance at 517 nm.
The ABTS decolorization assay was carried out using the protocol adapted to a microplate [20,21]. The ABTS●+ was prepared by mixing 7 mM ABTS (2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonate)) (Sigma-Aldrich®) solution (0.3602 g in 100 mL distilled water) with 2 mM potassium persulfate (2.45 µM, 0.0662 g in 100 mL distilled water). The mixture was kept in darkness for 12–16 h at room temperature. After that, the absorbance of ABTS●+ was adjusted to 0.70 at 734 nm. The sample (10 μL) and ABTS●+ (190 μL) were mixed, and after one minute, the absorbance was measured using a microplate reader (Multiskan GO, Thermo Scientific). The results for the DPPH and ABTS assays were expressed as mg of Trolox equivalents/g of dry weight (mgTE/gdw) using a calibration curve with 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox; 0.04–0.2 mg/mL; R2 = 0.98 and 0.99, respectively).
The FRAP (ferric reducing antioxidant power) assay was carried out according to Pulido et al. [22], adapted to a 96-well microplate. The FRAP reagent (0.3 M acetate buffer (pH 3.6), 10 mM 2,4,6-tri(2-pyridyl)-s-triazine (TTPZ; Sigma-Aldrich®) solution in 10 mM HCl, and 20 mM FeCl3.6H2O; 10:1:1 v/v) was prepared daily and incubated at 37 °C for 30 min. For the reaction, 6 µL of the sample, 18 µL of distilled water, and 180 µL of the FRAP reagent were mixed. The resulting solution was incubated at 37 °C for 60 min, and the absorbance was measured using a microplate reader (Multiskan GO, Thermo Scientific) at 595 nm. The results obtained were expressed as mg of Fe+2 equivalents/g of dry weight (mgFe+2/gdw), using a calibration curve with Fe2SO4 (Sigma-Aldrich®) in 80% methanol (0.1–1 mg/mL; R2 = 0.99).

2.6.3. Identification of Phenolic Compounds

The phenolic profile of the fermented extract was obtained by reverse-phase high-performance liquid chromatography/electrospray ionization/mass spectrometry (RP-HPLC/ESI/MS) according to [14], using a Varian HPLC system, including an autosampler (Varian ProStar 410, Palo Alto, CA, USA), a PDA detector (Varian ProStar 330, Palo Alto, CA, USA), and a ternary pump (Varian ProStar 230I, Palo Alto, CA, USA). The separation was carried out at 30 °C in a Denali C18 column (150 mm × 2.1 mm, 3 µm, Grace, Palo Alto, California, USA) in 5 µL samples. The mobile phase (wash phase) was methanol, and the eluents were formic acid (0.2%, v/v) and acetonitrile (solvent A and B, respectively). The elution gradient applied was: initial, 3% B; 0–5 min, 9% B linear; 5–15 min, 16% B linear; 14–45 min, 50% B linear. A mass spectrometer (Varian 500-MS IT Mass Spectrometer, Palo Alto, CA, USA) equipped with an electrospray ionization source, operated in negative mode [M-H]−1, was used for the LC-ESI-MS analysis. Data were collected and processed using the MS Workstation software (V 6.9). Samples were initially analyzed in full-scan mode, acquired in the m/z range of 50–2000.

2.7. Experimental Design

To select the best strain and time for the largest TPC content and AA, a completely randomized design with factorial arrangement was established. The factors evaluated were fungal strain (A. niger GH1, A. awamori, and A. niger HT4) and culture time (0, 12, 24, 36, 48, 60, and 72 h) in triplicate (n = 63).
After that, a 2k Box, Hunter, and Hunter (BHH) design was set up to identify the factors with a significant effect on the TPC extraction and AA. For this purpose, six factors (inoculum size, temperature, moisture, MgSO4, NaNO3, and K2HPO4) at two levels each were used to construct a condensed matrix with eight treatments (Table 1). All treatments were performed in triplicate and expressed as a mean (n = 3) ± standard deviation. Data were submitted to Analysis of Variance (ANOVA) and Tukey´s test using the Statistica 7.0 software (Stat Soft, Tulsa, OK, USA). A p-value ≤ 0.05 was considered to indicate a significant difference. Correlations between phenolic compound content (HP, CP, and TPC) and the activity in antioxidant assays (ABTS, DPPH, and FRAP) were determined using Pearson´s correlation coefficient (p < 0.01).

3. Results

3.1. Physicochemical Characterization of Cascalote Pods

The chemical composition of cascalote pods (Table 2) was determined in order to evaluate their nutritional feasibility to be used as a substrate for fungal growth in an SSF process. The main components of cascalote pods are carbohydrates and fiber, whereas proteins constitute both elemental carbon and nitrogen sources for fungal growth and enzyme production [15]. The physicochemical values for the water absorption capacity (WAC), critical humidity point (CHP), and maximum moisture of the substrate (see Table 2) indicate the potential of any material to be used as a substrate in an SSF process.

3.2. Kinetics of TPC Extraction and AA Using Aspergillus Strains

The TPC release and AA assays (DPPH, ABTS, and FRAP), in relation to fermentation time, are shown in Figure 1. All of the evaluated strains could use cascalote pods as a substrate-support for their growth. TPC release was fast at the beginning, reaching a maximal value after 12 h of fermentation for all of the strains. At 12 h, the amount of TPC released was 70.22, 102.21, and 117.69 mgGAE/gdw for A. niger HT4, A. awamori, and A. niger GH1, respectively. After that time, TPC started to decrease, attaining a concentration of 55 mgGAE/gdw for all of the strains. Similar patterns were observed in the AA assays, DPPH (Figure 1b), ABTS (Figure 1c), and FRAP (Figure 1d), which achieved the largest values at 12 h for all of the strains. Based on the obtained results, A. niger and 12 h of fermentation time were selected for further studies.
Data from the extraction kinetics were used to estimate the relation between the phenolic compounds and antioxidant activity of the fermented extracts on the cascalote pods through the Pearson correlation coefficient. As shown in Table 3, the phenolic compound content (HP, CP, and TPC) was significantly correlated with the in vitro antioxidant activity measured by ABTS, DPPH, and FRAP.

3.3. Significant Factors for TPC Release by SSF

Experimental design is a useful tool for defining the conditions necessary to be able to perform a minimal number of experiments. The BHH design was performed to identify the main factors that influence TPC release from cascalote pods by SSF. For that, an eight-treatment experimental matrix was constructed to evaluate three process parameters (inoculum size, temperature, and moisture) and three media components (KH2PO4, NaNO3, and MgSO4) in the hydrolyzed, condensed, and total polyphenol content, as well as the antioxidant activity against DPPH and ABTS radicals in the cascalote pods fermented with A. niger GH1; the results are shown in Table 4. Maximal values were obtained in treatment 6, without significant differences in comparison to treatment 7, which were 0.98-, 0.79-, and 1.1-fold higher than those obtained in treatment 2 for the TPC, ABTS, and DPPH values, respectively.
To evaluate the influence of these factors, the absolute values of standardized effects were estimated; their behaviors are shown in Figure 2. Moisture was the most influential (p < 0.05) and had a positive effect, indicating that as the factor level increased, the expected response also increased (Figure 2a). In contrast, TPC release was negatively affected by KH2PO4, MgSO4, and KCl concentrations (Figure 2b–d), revealing that an increase in these factors harms the TPC release. Temperature and NaNO3 showed no significant effect (p > 0.05). Therefore, any level of these factors could be used for evaluation. Media components that show a negative (KH2PO4, MgSO4, and KCl concentration) or non-significant (temperature and NaNO3) effect are recommended for use in low levels (see Table 1). Therefore, treatment 6 was selected due to the highest values of TPC release and higher activity in antioxidant assays (DPPH, ABTS, and FRAP).

3.4. Effect of SSF on Chemical Composition, Tannin Content, and Antioxidant Activity

The chemical composition, tannin content, and antioxidant activity of treatment 6 were determined and compared to control; no fermentation was conducted (Table 5). Significant changes were observed for all of the parameters (p ≤ 0.05), including an increase in moisture, fat, protein, and ash content. A reduction in the fiber and carbohydrate contents as an effect of SSF was observed. Concerning tannin content, SSF by A. niger GH1 increased the content of hydrolyzed, condensed, and total polyphenols, reaching values that were 66%, 337%, and 118%, respectively, higher than the control (without fermentation). The activity of the fermented extracts in the DPPH, ABTS, and FRAP assays was improved by 66.76%, 93%, and 55.67%, respectively, compared to the control.

3.5. Identification of Phenolic Compounds by RP-HPLC-ESI-MS

The extracts obtained at 0 and 12 h of SSF under the conditions of treatment 6 (see Table 1) were characterized by RP-HPLC-ESI-MS. A total of nine phenolic compounds were detected (Table 6), mainly belonging to the family of hydroxybenzoic acids and ellagitannins; however, two of them were unidentified according to the existing database, but according to the phenolic compound profiles and the weight of unidentified compounds, they are closely related to other ellagitannins. These compounds will be further elucidated using other procedures (i.e., Nuclear Magnetic Resonance).

4. Discussion

A culture medium is characterized by a mixture of nutrients that, in adequate concentrations and under optimal physical conditions, allows microbial growth and metabolic processes to occur [14]. The obtained results (Table 2) are in the range of the values reported in the literature for moisture (3%), protein (3.85–5.34%), ash (1.87–2.58%), fat (0.19–3.35%), and carbohydrate (71.62–88.82%) content [1,23].
In the SSF process, WAC, CHP, and maximum moisture are important parameters for the evaluation of the vegetal material’s suitability as a solid support for fungal growth and metabolite production. WAC indicates the amount of water that can be absorbed by the substrate, and it is related to the hydroxyl groups present on it and allows additional water interactions by hydrogen bonding [12,24]. Thus, a high WAC value is convenient for the SSF process because moisture content can be modified, thus allowing microbial growth. Cascalote pods presented a WAC value of 2.97 g gel/gdw, which is in the range reported (2.16–3.4 g gel/gdw) for agro-industrial byproducts considered as suitable supports for SSF such as grape waste [25], corn cobs [26], candelilla stalks [26], rambutan peels [12], and fig byproducts [27]. The CHP represents the water linked to the support that cannot be used for the biological functions of the microorganism. Microbial growth is promoted at low CHP values since high values affect it in a big proportion of the water linked to the substrate; therefore free water content is low [15]. The CHP of the cascalote pods was 3.75%, which is lower than those reported for fig byproducts (4.63%), sugarcane bagasse (9.46%), pomegranate peel (10.13%), coconut husk (16%), corn cobs (27%), grape waste (53.33%), pineapple waste (55.6%), and mango seeds (56.5%)—materials successfully used as substrate supports in SSF [11,14,15,25,26,27]. Additionally, cascalote pods could be used as a substrate in the SSF process at high moisture levels due to the maximum moisture level that they obtained (79.33%); however, it is recommended to work at a moisture level below 70% in order to avoid oxygen transfer limitations, particle agglomeration, and bacterial growth [12,28]. Based on their physicochemical characterization, cascalote pods are suitable for use as a substrate support for SSF.
Filamentous fungi are the most used microorganism in SSF, and they have great potential to release bioactive compounds from vegetal matrices [8]. Fungi can assimilate a wide variety of carbon sources, thus being able to synthesize, degrade, and transform several phenolic compounds and other aromatic compounds [29,30]. During SSF, fungi produce several enzymes that degrade the cell wall components, resulting in an enhanced phenolic compound extraction [15]. A. niger GH1 has been previously reported as a potential fungus for the degradation of lignocellulosic materials and the release of phenolic compounds [11,12,14]. In this study, the use of A. niger GH1 increased the TPC release 1.67-fold and the AA 2.17-fold as compared to the other Aspergillus strains evaluated.
The TPC release was positively correlated with AA (0.95; p < 0.01), confirming that the increase in AA values was due to the increase in TPC release during the SSF process. This correlation is in accordance with the previous studies published by Paz-Arteaga et al. [15], Jericó-Santos et al. [31], and Buenrostro-Figueroa et al. [14] on the SSF of pineapple, tamarind, and pomegranate byproducts, which reported correlation values between TPC and DPPH of 0.63, 072, and 0.86, respectively. Increments in the TPC released are associated with the fungal enzymes produced during the microbial growth phase. These enzymes (xylanases, pectinases, proteases, and glucosidase, among others) are responsible for the oxidative degradation of lignin and the breakdown of the links between the cell wall matrix and phenolic compounds, which results in their release [15,32].
Exploring the effects of nutritional and physical parameters in SSF influences microbial growth and their metabolic processes. Moisture plays an important role in fungal growth. SSF requires the close control of water content; depending on the microbial strain used, a specific moisture content is needed to ensure growth and metabolite production [33]. In this study, TPC release was favored at high levels of moisture (60%), similar to the results obtained by Buenrostro-Figueroa et al. [27] in the SSF of fig byproducts using A. niger GH1.
Magnesium is necessary for fungal nutrition, and it has an important role in hyphae development. An optimal concentration of this mineral improves the sporulation rate, thus promoting an efficient enzyme synthesis and consequently, fungal biomass proliferation and phenolic compound release [14,34]. Using A. niger GH1 and pomegranate husk to obtain ellagic acid and total phenolic compounds by SSF, Sepulveda et al. [34] and Buenrostro-Figueroa et al. [14] found that increments in MgSO4 levels improved the release of ellagic acid and total phenolic compounds, respectively. Furthermore, those authors reported that the best phenolic compound release was attained at high levels of KH2PO4 and KCl. In the present study, the same KH2PO4, KCl, and MgSO4 levels were used; however, a contrary effect was observed. This may be due to the substrate support (cascalote pods) itself containing sufficient amounts of minerals (Mg, P, and K) needed for both microbial growth and TPC release. The addition of higher amounts of minerals could have affected the enzyme production [35]. The cell wall of A. niger contains carbohydrates, proteins, ash, and lipids [36]. Increases in protein and lipid content are related to the fungal biomass present in fermented cascalote pods. During fungal growth, the available nutrients are used to synthesize lipids as mycelium components. Reduction in carbohydrate and fiber contents are associated with fungal growth since these components are used by the fungi as a carbon source for its development and production of cell wall-degrading enzymes [15,32].
Out of all the treatments evaluated, the best values for the response variables (TPC and AA) were obtained in treatment 6, which may have been due to these conditions (30 °C, 60% moisture, and a concentration of mineral salts in the medium (g/L) of: KH2PO4: 1.52, NaNO3: 7.65, MgSO4•7H2O: 1.52, and KCl: 1.52) promoting the better growth and metabolic processes of A. niger GH1. SSF substantially improved the amount of phenolic compounds released from cascalote pods as well as the antioxidant activity. This is explained by the fact that several enzymes with important hydrolytic activities participate in SSF, inducing the release of phenolic compounds from polymeric matrices [15,37].
Based on the above results, SSF was successfully carried out by A. niger GH1 on cascalote pods, with 12 h as the best time for TPC release. This is the first report to be conducted for cascalote pods under the conditions described. Using extraction by maceration with methanol, Sánchez et al. [38] reported values of 21.71 mg/gdw for condensed tannins and 32.06 mg/gdw for total tannins. Rojas-Morales et al. [39] reported values for condensed and total tannins of 13 and 34 mg/gdw, respectively. A total condensed tannins content of 7.72 mg/gdw was reported by Pineda-Peña et al. [40]. The values for condensed and total polyphenols released from cascalote pods in the present work are 120–521% and 265–288% higher than the values previously reported [38,39,40].
In addition, the cascalote pods exhibited strong antioxidant activity by reducing the agents for ferric ions and scavenging free radicals. According to the ABTS assay, SSF provided an increase of up to 93% in the AA of the extract (498.46 mgTE/gdw) as compared to the value before SSF (258.18 mgTE/gdw). Ethanolic extract from cascalote was evaluated against DPPH and ABTS antioxidant assays [41]. At 500 mg/L, the inhibition rate was higher than 90% in both cases. Based on the strongly positive and highly significant correlation between phenolic compound content and activity in antioxidant assays (DPPH, ABTS, and FRAP), the increase in AA can be attributed to the amount and type of phenolic compounds released by the SSF. These results show the feasibility of obtaining TPC with AA from vegetal matrices via SSF in comparison to chemical processes or the use of commercial enzymes.
The RP-HPLC-ESI-MS of cascalote extracts showed that the main compounds were 5-O-galloylquinic acid, ellagic acid derivatives, corilagin, gallagyl-hexoside, lagerstannin B derivative, geraniin, and ellagic acid. There have been no reports indicating the identification of phenolic compounds obtained from cascalote pods by SSF; however, some of these compounds have been previously reported from the hydroalcoholic extracts of C. coriaria [40,42]. In addition, other compounds, such as ethyl gallate, methyl gallate, gallic acid, tetragalloylglucose, pentagalloylglucose, valoneic acid dilactone, digalloylshikimic acid, and other phenolic compound derivatives, have been cited [2,4,43].
From the nine compounds detected, seven of them increased in concentration at 12 h of SSF (in terms of units of absorbance), with 5-o-galloyquinic acid, corilagin, geraniin, and ellagic acid having values 12.19-, 2.34-, 5.11-, and 7.72-fold higher than those obtained at 0 h of SSF. The lagerstannin B derivative was only found at 12 h of SSF, while gallagyl-hexoside was only detected at 0 h. The differences between the phenolic profile and absorbance values might be due to the phenolic compounds being in free or conjugated form (esterified). During SSF, these bonds are broken by the action of microbial enzymes, which facilitates the partial or complete release of the phenolic compounds, therefore improving their solubility or producing new molecules [15].
Identified polyphenol molecules in cascalote pods have different biological activities, such as antioxidant effects, hepatoprotective effects [41], anthelmintic effects [39,44], arginase inhibitory activity [45], antimicrobial activity [4,43,46], and gastroprotective effects [40]. These benefits provide a wide range of possible applications in the food, pharmaceutical, and cosmetic industries.
Accurate data related to cascalote production were not found; however, a yield of 150 kg of pods per tree has been reported [1]. Using the bioprocess described here, up to 18.6 kg of TPC per tree of cascalote pods could be obtained (124 kg of TPC/ton). Considering the commercial prices on the Sigma-Aldrich website ( accessed on 31 August 2023) for corilagin (SKU: 75251), geraniin (SKU: PHL80994), and ellagic acid (SKU: PHL89653) (594 USD/10 mg, 501 USD/10 mg, and 271 USD/50 mg, respectively), the SSF extraction process represents a profitable and sustainable alternative for the acquisition of valuable compounds with industrial applications. These results confirm that SSF permits the recovery of larger amounts of high-value molecules through a process that involves a shorter amount of time. With particular focus on the highly desired circular economy model, an alternative to the diversification of cascalote pods has thus been reported.

5. Conclusions

A. niger GH1 utilized cascalote pods as a carbon source and released phenolic compounds with antioxidant activity. The factors that most influenced the TPC release were moisture and KH2PO4 concentration. The higher TPC release and antioxidant activity were obtained at the following SSF conditions: A. niger GH1 (1 × 107 sp/gdw) at 12 h of culture, 30 °C, 60% moisture, and a concentration of mineral salts in the medium (g/L) of: KH2PO4: 1.52, NaNO3: 7.65, MgSO4•7H2O: 1.52, and KCl: 1.52. Corilagin, lagerstannin, geraniin, and ellagic acid were the main phenolic compounds identified by RP-HPLC-ESI-MS in the cascalote extracts. The development of a bioprocess such as the SSF-assisted extraction conducted in this study allowed for the significant increase in TPC release and exhibited high AA, thus offering an alternative use for this underutilized material—the cascalote pods. The recovered molecules are of great interest in the cosmetic, pharmacy, and food industries and are considered to be high added-value products.

Author Contributions

F.L.-C.: performed lab research, methodology, and formal analysis. E.O.-R.: validation, formal analysis, and investigation. R.B.-J.: visualization, supervision, review and editing. J.C.T.-A.: conceptualization, resources, visualization, supervision, writing, review and editing. J.A.A.-V.: RP-HPLC-ESI-MS, investigation, review and editing. J.J.B.-F.: conceptualization, software, formal analysis, resources, supervision, project administration, writing—original draft, writing—review and editing, funding acquisition. All authors have read and agreed to the published version of the manuscript.


This research received no external funding.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be made available upon request.


The authors are grateful for the support provided by the Centro de Investigación en Alimentación y Desarrollo (CIAD), Departamento de Investigación en Alimentos (DIA-UAdeC), and Universidad Autónoma Agraria Antonio Narro (UAAAN).

Conflicts of Interest

The authors declare no conflict of interest.


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Figure 1. Kinetics of: (a) total polyphenol content, (b) antioxidant activity by DPPH assay, (c) antioxidant activity by ABTS assay, and (d) antioxidant activity by FRAP assay, for the fermented extracts of Aspergillus strains on cascalote pods.
Figure 1. Kinetics of: (a) total polyphenol content, (b) antioxidant activity by DPPH assay, (c) antioxidant activity by ABTS assay, and (d) antioxidant activity by FRAP assay, for the fermented extracts of Aspergillus strains on cascalote pods.
Fermentation 09 00823 g001
Figure 2. Effect of the significant factors (a) Moisture, (b) KH2PO4, (c) MgSO4 and (d) KCl on TPC release by SSF with A. niger GH1.
Figure 2. Effect of the significant factors (a) Moisture, (b) KH2PO4, (c) MgSO4 and (d) KCl on TPC release by SSF with A. niger GH1.
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Table 1. Treatment matrix of the experimental design by Box, Hunter, and Hunter.
Table 1. Treatment matrix of the experimental design by Box, Hunter, and Hunter.
CodeFactorLow level (−1)High level (1)
AInoculum (spores/gdw)1 × 1061 × 107
BTemperature (°C)3040
CMoisture (%)5060
DKH2PO4 (g/L)1.523.04
ENaNO3 (g/L)3.047.65
FMgSO4•7H2O (g/L)1.523.04
GKCl (g/L)1.523.04
Table 2. Physicochemical characterization of cascalote pods.
Table 2. Physicochemical characterization of cascalote pods.
Moisture (% w/w)3.36 ± 0.15
Fat (g/100 gdw)0.65 ± 0.09
Fiber (g/100 gdw)6.54 ± 0.25
Protein (g/100 gdw)3.44 ± 0.13
Ash (g/100 gdw)2.13 ± 0.21
Carbohydrates (g/100 gdw)87.24 ± 0.96
Water absorption capacity (g of gel/gdw)2.97 ± 0.07
Critical humidity point (%)3.75 ± 0.29
Maximum moisture of cascalote pods (%)79.33 ± 2.08
gdw: gram of dry weight.
Table 3. Pearson’s correlation coefficients between phenolic compounds (HP, CP, and TPC) and activity in antioxidant assays (ABTS, DPPH, and FRAP).
Table 3. Pearson’s correlation coefficients between phenolic compounds (HP, CP, and TPC) and activity in antioxidant assays (ABTS, DPPH, and FRAP).
HP10.94 *0.98 *0.94 *0.94 *0.82 *
CP 10.98 *0.93 *0.94 *0.84 *
TPC 10.95 *0.95 *0.85 *
ABTS 10.95 *0.87 *
DPPH 10.95 *
* Significant correlations (p ˂ 0.01).
Table 4. Treatment matrix of the experimental design by Box, Hunter, and Hunter used to determine the influence of independent factors (A, B, C, D, E, F, and G) on the TPC and AA (ABTS, DPPH, FRAP) of extracts obtained by SSF with A. niger GH1 on the cascalote pods.
Table 4. Treatment matrix of the experimental design by Box, Hunter, and Hunter used to determine the influence of independent factors (A, B, C, D, E, F, and G) on the TPC and AA (ABTS, DPPH, FRAP) of extracts obtained by SSF with A. niger GH1 on the cascalote pods.
TreatmentABCDEFGTPC (mg/gdw)ABTS (mgTE/gdw)DPPH (mgTE/gdw)FRAP (mgFE+2/gdw)
1−1−1−1111−171.62 ± 2.23 f271.46 ± 3.15 de237.32 ± 5.51 f735.78 ± 12.85 d
21−1−1−1−11162.95 ± 3.51 g247.90 ± 15.01 e226.25 ± 7.39 f737.49 ± 15.09 d
3−11−1−11−1199.88 ± 1.96 c380.68 ± 19.01 b365.92 ± 4.54 c945.92 ± 55.67 c
411−11−1−1−179.48 ± 0.52 e294.85 ± 8.28 cd297.58 ± 6.97 e800.07 ± 53.24 d
5−1−111−1−1192.55 ± 1.45 d384.55 ± 15.28 b345.96 ± 9.63 cd925.42 ± 39.09 c
61−11−11−1−1124.17 ± 1.94 a447.64 ± 5.81 a498.46 ± 13.15 a1251.13 ± 29.45 a
7−111−1−11−1114.23 ± 2.67 b438.81 ± 14.00 a452.64 ± 3.94 b1121.07 ± 44.45 b
8111111171.84 ± 2.78 f320.19 ± 14.71 c329.10 ± 15.32 d1019 ± 52.78 bc
CodeFactorLow level (−1)High level (1)
AInoculum (spores/gdw)1 × 1061 × 107
BTemperature (°C)3040
CMoisture (%)5060
DKH2PO4 (g/L)1.523.04
ENaNO3 (g/L)3.047.65
FMgSO4•7H2O (g/L)1.523.04
GKCl (g/L)1.523.04
Different lowercase letters indicate no significant differences among treatments (Tukey’s test; p ≤ 0.05).
Table 5. The SSF of cascalote pods (mixed with vermiculite; 50:50 w/w) using A. niger GH1 and its effect on chemical composition, tannin content, and antioxidant activity.
Table 5. The SSF of cascalote pods (mixed with vermiculite; 50:50 w/w) using A. niger GH1 and its effect on chemical composition, tannin content, and antioxidant activity.
Parameter/TreatmentControl (0 h)Treatment 6 (12 h)
Moisture (g/100 gdw) 4.90 ± 0.04 b5.40 ± 0.20 a
Fat (g/100 gdw)0.23 ± 0.05 b0.34 ± 0.10 a
Fiber (g/100 gdw)5.54 ± 0.23 a1.77 ± 0.12 b
Protein (g/100 gdw)3.24 ± 0.15 b3.59 ± 0.10 a
Ash (g/100 gdw)52.88 ± 0.95 b59.28 ± 0.93 a
Carbohydrates (g/100 gdw)38.11 ± 1.95 a35.02 ± 1.76 b
Hydrolyzed polyphenols (mgGAE/gdw)45.76 b76.22 a
Condensed polyphenols (mgCE/gdw)10.97 b47.95 a
Total polyphenol content (mg/gdw)56.73 b124.17 a
Antioxidant activity:
DPPH (mgTE/gdw)266.63 b444.64 a
ABTS (mgTE/gdw)258.18 b498.46 a
FRAP (mgFe+2/gdw)354.03 b551.13 a
Different letters indicate no significant differences between treatments (Tukey α = 0.05).
Table 6. Phenolic compound profiles on cascalote pods fermented by A. niger GH1 and identified using the RP-HPLC-ESI-MS technique.
Table 6. Phenolic compound profiles on cascalote pods fermented by A. niger GH1 and identified using the RP-HPLC-ESI-MS technique.
#RT[M-H]Bioactive CompoundMolecular FormulaFamily0 h12 h SSF
110.41342.55-O-Galloylquinic acidC14H16O10Hydroxybenzoic acids0.0260.343
215.02798.4Ellagic acid derivate Hydroxybenzoic acids0.1361.539
317.64494.7Unidentified 0.3732.479
418.511118.1Unidentified 0.3842.451
724.62968.1Lagerstannin B derivate EllagitanninsND2.458
928.84300.6Ellagic acidC14H6O8Hydroxybenzoic acid dimers0.2472.155
RT = retention time; UA = unit of absorbance; ND = not detected.
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López-Cárdenas, F.; Ochoa-Reyes, E.; Baeza-Jiménez, R.; Tafolla-Arellano, J.C.; Ascacio-Valdés, J.A.; Buenrostro-Figueroa, J.J. Solid-State Fermentation as a Sustainable Tool for Extracting Phenolic Compounds from Cascalote Pods. Fermentation 2023, 9, 823.

AMA Style

López-Cárdenas F, Ochoa-Reyes E, Baeza-Jiménez R, Tafolla-Arellano JC, Ascacio-Valdés JA, Buenrostro-Figueroa JJ. Solid-State Fermentation as a Sustainable Tool for Extracting Phenolic Compounds from Cascalote Pods. Fermentation. 2023; 9(9):823.

Chicago/Turabian Style

López-Cárdenas, Francisco, Emilio Ochoa-Reyes, Ramiro Baeza-Jiménez, Julio C. Tafolla-Arellano, Juan A. Ascacio-Valdés, and José J. Buenrostro-Figueroa. 2023. "Solid-State Fermentation as a Sustainable Tool for Extracting Phenolic Compounds from Cascalote Pods" Fermentation 9, no. 9: 823.

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