Bioproduction of Nordihydroguaiaretic and Ellagic Acid from Creosote Bush Leaves (Larrea tridentata) Using Solid-State Fermentation with Aspergillus niger GH1

Abstract

Creosote bush (Larrea tridentata), a shrub distributed across approximately 19 Mha of arid North American regions, has traditional applications in folk medicine due to the presence of bioactive molecules such as nordihydroguaiaretic acid (NDGA) and ellagic acid (EA). This study investigated the implementation of a solid-state fermentation (SSF) optimization process employing creosote bush leaves as substrate using Aspergillus niger GH1 to improve NDGA and EA extraction. This study was based on previous research by our group that identified key parameters for NDGA production in a related SSF system. Creosote bush is a recognized source of these bioactive compounds, which possess antioxidant and anti-inflammatory properties. Conventional extraction methods often exhibit limitations in efficiency and sustainability. The efficacy of A. niger GH1 in SSF has been previously established with diverse substrates. In this study, A. niger GH1 was employed in an SSF process utilizing creosote bush leaves as a substrate using a Box–Behnken experimental design. The accumulation of NDGA and EA, which were quantified by HPLC-MS, yielded values of 1.20 ± 0.32 mg g⁻¹ for EA and 7.39 ± 0.52 mg g⁻¹ for NDGA. In vitro antioxidant assays (DPPH and ABTS) demonstrated significant antioxidant activity, with inhibition percentages of 55.69% and 84.84%, respectively. These results indicate that A. niger GH1-mediated SSF using Creosote bush leaves is a viable and sustainable strategy for producing these valuable bioactive compounds.

1. Introduction

Ellagic acid (EA) is a secondary metabolite widely distributed in the plant kingdom, often found in fruits and vegetables, many of which have a history of use in folk medicine. It occurs in both free form and as a constituent of more complex molecules, notably ellagitannins. These complex forms can be metabolized, yielding EA and other compounds, including sugars [1]. Structurally, EA is a dilactone of hexahydroxydiphenic acid (HHDP) as shown in Figure 1, a dimeric derivative of gallic acid, and is primarily generated through the hydrolysis of ellagitannins. EA has garnered significant research interest due to its reported antioxidant, anti-inflammatory, antimutagenic, and antiproliferative properties [2]. On the other hand, nordihydroguaiaretic acid (NDGA), as shown in Figure 2, is a compound that belongs to the lignan family, which are diphenolic compounds present in various plant species that can be linked by ester, lactone, or carbon-carbon bonds [3]. NDGA possesses different biological activities, such as antioxidants, antimicrobial, anticancer, anti-inflammatory, and may even inhibit some enzymes, such as lipooxygenases [4,5,6].

The extraction of phenolic compounds presents a significant challenge. Conventional methods, such as maceration, hydro-distillation, and infusion, rely on differential solubilization in various solvents, with extraction efficiency often modulated by temperature, agitation, and time [7]. While simple to implement, these techniques are slow and inefficient, potentially degrading bioactive compounds and typically requiring large volumes of organic solvents [8]. The extraction of bioactive compounds should prioritize compound quality and functionality without compromising efficiency, cost-effectiveness, and sustainability [9]. In this context, SSF has emerged as a promising biotechnological alternative. SSF offers sustainable and cost-effective bioprocesses, enabling the direct utilization of undervalued plants and/or agro-industrial wastes to produce high-value compounds. Although the SSF process offers several advantages, it also presents significant challenges, the main ones being the scaling and control of operational parameters such as pH, temperature, and oxygen gradients within the bioreactor [10]. In this bioprocess, filamentous fungi, particularly those within the Basidiomycota and Ascomycota subdivisions, are widely employed due to their inherent physiological, biochemical, and enzymatic properties [11]. In addition, this bioprocess has demonstrated its potential for producing bioactive compounds, including polyphenols, enzymes, antibiotics, and biofuels [12]. The global imperative is to shift from a linear, fossil resource-dependent economy to a circular model valorizing low-value lignocellulosic waste. Annually, 181.5 billion tons of lignocellulosic biomass are produced globally; however, only 8.2 billion tons are currently utilized, representing a substantial untapped resource for diverse applications, such as the production of valuable compounds through enzymatic processes [13,14]. Plant biomass, formed by complex polysaccharides, presents a challenge for microbial enzymatic degradation. To improve enzymatic hydrolysis, pretreatments of the plant material are frequently used, including physical methods such as particle size reduction (milling), ultrasound, and microwaves, alongside chemical treatments. These pretreatments aid in disrupting the plant cell wall, thus enhancing hydrolysis efficiency. However, careful selection of the pretreatment method is essential to avoid the formation of nondesirable by-products during lignocellulosic biomass processing [15]. The creosote bush, an undervalued perennial shrub native to the arid regions of North America, is distributed across approximately 19 Mha of the continent, specifically in central and northern Mexico and the southern United States [16], is recognized for its robust survival in harsh environments [17]. This resilience is attributed to its rich phytochemical profile, featuring bioactive compounds. Historically, creosote bush has been employed in folk medicine, reflecting its potent biological activities, including antioxidant, antimicrobial, and antiviral properties [18,19]. Furthermore, this extensive distribution, coupled with the fact that approximately 50% of its leaf dry weight is extractable matter, positions the creosote bush as a valuable source for applications in various industries, particularly in the development of functional ingredients and therapeutic agents. The fungal strain Aspergillus niger GH1, isolated from the creosote bush in the Mexican desert [20], is extensively utilized in SSF to produce diverse industrial enzymes, including cellulases [21], proteases [22], and invertase [23], demonstrating efficacy in the bioconversion of various agro-industrial residues. Examples include pineapple waste utilization [24], polyphenol extraction from mango seeds [25], and bioactive compound recovery from grape pomace [26]. This study aimed at the implementation of an SSF optimization process employing creosote bush leaves as a substrate to produce NDGA and EA, building on previous research by our group that identified key parameters for NDGA production in a related SSF system. This approach is particularly relevant considering the limited recent literature concerning the application of SSF to creosote bush biomass.

2. Materials and Methods

2.1. Raw Material Pretreatment

Creosote bush was collected from Saltillo, Coahuila (25°33′18.3″ N 100°55′34.7″ W). The plant material was disinfected in a 10% (v/v) water-sodium hypochlorite (NaOCl) solution. Subsequently, it was dried at 50 °C for 72 h. Leaves and flowers were manually removed and ground to a particle size less than 2 mm. The ground plant material was stored in plastic bags protected from light at room temperature.

2.2. Physicochemical Characterization of Creosote Bush

To determine water absorption capacity (WAC), a modified version of the method described by Islam et al. (2024) [27] was employed. Raw plant material (1 g) was placed in a 30 mL centrifuge tube and mixed with 9 mL of distilled water. The mixture was incubated at 30 °C and manually stirred for 10 min. Then, the samples were centrifuged (model 80-2B jfLAREN, Guamuchil, Sinaloa, Mexico) at 3000 rpm for 30 min, after the centrifugation process, the supernatant was discarded, and the remaining “gel” was weighed. WAC was calculated and expressed as gel grams per gram of dry weight (gel-g g⁻¹).

$$WAC={\displaystyle \frac{Weigth of gel \left(\mathrm{g}\right)}{weigth of dry sample \left(\mathrm{g}\right)}}$$

(1)

The moisture content and dry matter content of the plant material were determined gravimetrically. One gram of the sample was weighed using a thermobalance (OHAUS, Parsippany, NJ, USA). By combining the WAC values with the moisture and dry matter content, the maximum moisture content that the substrate can support was calculated [28].

$$\begin{gathered} General balance=M1+M2=M3 \\ Solids balance=M1\left(Xs,1\right)=M3\left(Xs,3\right) \\ Xs,3=\left(M1-{\displaystyle \frac{\left(M1\right)\left(Xs,1/100\right)}{M3}}\right)\ast 100 \end{gathered}$$

(2)

where Xs,3 is the maximum moisture content that the substrate can support (%), M1 is the mass of dry material (g), Xs,1 is the percentage of total solids divided by 100, and M3 is the value of WAC.

To determine the total sugars, the samples were previously hydrolyzed; 10 mg of each sample was hydrolyzed with 2 mL of concentrated (96% v/v) H₂SO₄ for 3 h. Following hydrolysis, the resulting liquid was diluted with distilled water, filtered through Whatman No. 1 paper, and the filtrate was adjusted to a final volume of 50 mL with distilled water in a volumetric flask. For the quantification of total sugars, the method of He et al. (2021) [29] was followed with slight modifications. Dextrose was employed as a standard, ranging from 0 to 500 ppm. A 250 µL aliquot of each sample was mixed with 500 µL of Anthrone reagent and incubated in an ice bath for 5 min. The mixture was then heated in a water bath at 80 °C for 15 min, followed by another 5 min incubation in an ice bath. Finally, 200 µL of each sample was transferred to a microplate reader Multiskan SkyHigh (Thermo Fisher Scientific, Waltham, MA, USA), and the absorbance was measured at 530 nm.

The proximate analyses were performed as follows. The ash content was determined by incineration in a muffle (Thermo Scientific, Thermolyne, F48015-60, Waltham, MA, USA) at 500 °C. The lipid determination was carried out by gravimetry using the Soxhlet method [30]. Briefly, 50 mL of hexane were used as solvent, 4 g of sample were deposited on dry Whatman No. 1 filter papers at constant weight, and lipid extraction was carried out for 6 h with a Soxhlet apparatus. After extraction and recovery of the solvent by distillation, the residue was dried at 40 °C for 48 h. The lipid content was determined by gravimetric analysis, comparing the weight of the filter paper before and after the process according to the following equation:

$$Lipids\%={\displaystyle \frac{Wp1 \left(\mathrm{g}\right)+Smpl \left(\mathrm{g}\right)-Wp2 \left(\mathrm{g}\right)}{Smpl \left(\mathrm{g}\right)}}\times 100$$

(3)

where Wp1 represents the weight of dry Whatman filter paper before Soxhlet extraction, Smpl represents the weight of the dry sample, and Wp2 represents the weight of the Whatman filter paper after the Soxhlet extraction.

The fiber content was measured by the AOAC 962.09 method [31]. Briefly, a 0.5 g defatted sample obtained in the lipid determination step was sequentially hydrolyzed in 200 mL beakers with 1.25% (v/v) H₂SO₄ and 1.25% (v/v) NaOH at 100 °C for 30 min each. After each hydrolysis, the residue was filtered through dry Whatman No. 1 filter paper at constant weight and rinsed twice with hot distilled water. The final residue was dried at 40 °C for 48 h, and fiber content was determined gravimetrically using the following equation:

$$Fiber\%={\displaystyle \frac{Wf-Wi}{Sw}}\times 100$$

(4)

where Wf represents the final weight (g) of dry Whatman filter paper with sample after the determination, Wi represents the initial weight (g) of the dry Whatman filter paper at constant weight, and Sw represents the weight (g) of the sample used.

The level of crude protein was determined by the micro Kjeldahl method [32] with a conversion factor of 6.25. All measurements were conducted in triplicate.

2.3. Micro-Organism and Culture Medium

The strain A. niger GH1 was used from the collection of the DIA UAdeC, deposited in the mycological library of the University of Minho with the number MUM:23.16. The strain was conserved in a cryoprotective solution at −55 °C (skimmed milk/glycerol 9:1 v/v). The strain was reactivated in potato dextrose agar (PDA-Bioxon) for 5 d at 30 °C.

2.4. SSF Conditions and Bioactive Compounds Recovery (Box–Behnken Design)

A Box–Behnken experimental design was carried out as shown in

Table 1. Condensed experimental matrix and evaluated factors of the Box–Behnken experimental design.

Treatment	pH		MgSO4
1	−1		−1
2	−1		0
3	−1		1
4	0		−1
5	0		0
6	0		1
7	1		−1
8	1		0
9	1		1
Factor	Levels
	−1	0	1
pH	4.5	5	5.5
MgSO4 (g L−1)	0.26	0.76	1.26

All treatments were performed in triplicate.

. SSF was carried out with A. niger GH1 strain, 3 g of plant material was placed in sterile Petri dishes, and 7 mL of culture medium (distilled water, MgSO₄ concentrations and pH values varied according to treatment as shown in Table 1) with inoculum (1 × 10⁶ spores g⁻¹) was added to obtain a moisture of 70%, followed by the incubation at 30 °C for a final time of 48 h. Sampling was performed after 48 h by manual pressing to obtain a polyphenolic extract with 14 mL of a 50:50 (v/v) ethanol-water solution.

Fermentation extracts, generated according to a Box–Behnken experimental design, were subjected to quantitative analysis to determine the concentrations of hydrolyzable and condensed tannins. Hydrolyzable tannin content in the fermentation extracts of creosote bush leaves was determined using the Folin-Ciocalteu method, adapted from Noce et al. (2021) [33]. Briefly, 20 µL of the sample (1:100 v/v) was mixed with 20 µL of Folin-Ciocalteu reagent and incubated for 5 min. Subsequently, 20 µL of 0.01 M Na₂CO₃ and 125 µL of distilled water were added. A gallic acid standard curve (0–200 ppm) was used to quantify the tannin content. Absorbance was measured at 790 nm using a Multiskan SkyHigh microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). All measurements were performed in triplicate, and results were expressed as milligrams of gallic acid equivalents per gram of sample (mg-GAE g⁻¹). Condensed tannins were quantified using the methodology of HCl-Butanol, adapted from Palacios et al. (2021) [34] with some modifications. Catechin was employed as a standard, ranging from 0 to 1000 ppm. A 500 µL aliquot of each sample (1:50 v/v) was mixed with 1.5 mL of HCl-butanol solution (1:9 v/v) and 50 µL of ferric reagent. The resulting mixtures were incubated in a water bath at 100 °C for 1 h, and subsequently cooled to room temperature. A 250 µL volume of each sample was transferred to a 96-well microplate, and finally the absorbance was measured at 460 nm using a microplate reader Multiskan SkyHigh (Thermo Fisher Scientific, Waltham, MA, USA). All measurements were performed in triplicate, and the results were expressed as milligrams of catechin equivalents per gram of sample (mg-CE g⁻¹).

2.5. HPLC-ESI-MS Analysis

A Varian HPLC-MS system, comprising an autosampler (Varian ProStar 410), a ternary pump (Varian ProStar 230I), a PDA detector (Varian ProStar 330), and a liquid chromatograph ion trap mass spectrometer (Varian 500-MS IT Mass Spectrometer), all sourced from Palo Alto, CA, USA, was employed to identify the primary constituents within the fermentation extracts of creosote bush leaves. Prior to analysis, the extracts were filtered through a 0.45 µm nylon membrane. Samples (10 µL) were injected onto a Denali C18 column (150 × 2.1 mm, 3 µm, Grace, Palo Alto, CA, USA). The oven temperature was maintained at 30 °C. The eluents were formic acid (0.2%, v/v; solvent A) and acetonitrile (solvent B). The following gradient was applied: initial, 3% B; 0–5 min, 9% B linear; 5–15 min, 16% B linear; 15–45 min, 50% B linear. The column was then washed and reconditioned. The flow rate was maintained at 0.2 mL min⁻¹, and elution was monitored at 254, 280, 320, and 550 nm. The whole effluent (0.2 mL min⁻¹) was injected into the source of the mass spectrometer without splitting. All MS experiments were carried out in the negative mode [M-H]⁻¹. Nitrogen was used as nebulizing gas and helium as damping gas. The ion source parameters were spraying voltage 5.0 kV, and capillary voltage and temperature were 90.0 V and 350 °C, respectively. Data was collected and processed using MS Workstation software (V 6.9). Samples were first analyzed in full scan mode acquired in the m/z range 50–2000 [35].

The quantification of NDGA and EA was performed using a calibration curve (0–1000 ppm) for both standards.

2.6. Antioxidant Activity

The DPPH free radical scavenging activity was determined using a method described by Ordoñez-Torres et al. (2021) [36] with some modifications. A 60-μM DPPH solution was prepared in absolute ethanol. To initiate the reaction, 2.9 mL of this DPPH solution was mixed with 0.1 mL of the sample (1:50 v/v). The mixture was incubated for 30 min at room temperature in the dark. Subsequently, the absorbance of the samples was measured at 517 nm using Multiskan SkyHigh (Thermo Fisher Scientific, Waltham, MA, USA). DPPH-ethanol solution served as the control. The results were expressed as the percentage inhibition of DPPH radical.

$$\mathrm{DPPH} \mathrm{inhibition}\%={\displaystyle \frac{Ac-As}{Ac}}\times 100$$

(5)

where Ac represents the control absorbance of DPPH + ethanol and As represents the sample absorbance.

The ABTS radical scavenging assay was performed with the method described by López-Cardenas et al. (2023) [37] with some modifications. The ABTS⁺ radical was generated by combining 5 mL of 2.45 mM potassium persulfate with 5 mL of 7 mM ABTS solution. The mixture was incubated in the dark at room temperature for 12–16 h. Subsequently, the absorbance at 734 nm was measured, and the solution was diluted with ethanol to achieve an absorbance of 0.700. For the assay, 10 μL of each sample (1:50 v/v) was mixed with 1 mL of the adjusted ABTS⁺ solution, and the mixture was incubated for 1 min in the dark. Subsequently, the absorbance of the samples was measured at 734 nm using a Biomate 3 spectrophotometer (Thermo Spectronic, Madison, WI, USA). The results were expressed as the percentage inhibition of ABTS radical.

$$\mathrm{ABTS} \mathrm{inhibition}\%={\displaystyle \frac{Ac-As}{Ac}}\times 100$$

(6)

where Ac represents the control absorbance of ABTS + ethanol and As represents the sample absorbance.

2.7. Statistical Analysis

For the evaluation of the effect of pH and the concentration of MgSO₄ (g L⁻¹) of fermentation treatments, an experimental matrix of Box–Behnken was constructed using STATISTICA 7.0 software (Stat Soft, Tulsa, OK, USA) and comparison of means was performed via Tukey’s test (p < 0.05) using Oring Pro 2024 (Learning edition). All experiments were performed in triplicate, and the results were expressed as mean ± standard deviation and subjected to analysis of variance (ANOVA, p < 0.05).

3. Results

3.1. Physicochemical Properties of Creosote Bush Leaves

Creosote bush leaves exhibited the following characteristics: water absorption capacity (WAC) of 3.62 ± 0.02, moisture content of 5.00 ± 0.00%, and a maximum moisture capacity of 73.75 ± 0.01%. Proximal chemical analysis revealed the following composition: fat 5.11 ± 0.08%, fiber 16.82 ± 3.47%, protein 1.28 ± 0.01%, total sugars 37.06 ± 0.09%, and ash 9.41 ± 0.87% (

Table 2. Physicochemical characterization of creosote bush leaves (dry basis).

Parameters	Results
Moisture (%)	5.00 ± 0.00
Solids (%)	95.00 ± 0.00
Water absorption capacity (g of gel/g of dry weight)	3.62 ± 0.02
Maximum moisture of the support/substrate (%)	73.76 ± 0.01
Fat content (%)	5.11 ± 0.08
Fiber content (%)	16.82 ± 3.47
Protein content (%)	1.28 ± 0.01
Total sugar content (%)	37.06 ± 0.09
Ash content (%)	9.41 ± 0.87

).

3.2. Solid State Fermentation Conditions and Bioactive Compound Recovery (Box–Behnken Design)

The results of the Box–Behnken experimental design matrix are shown in Figure 3, illustrating the accumulation of hydrolyzable and condensed tannins. It was found that treatment 8 accumulated a higher concentration of hydrolyzable tannins (HT) and condensed tannins (CT) (36.17 ± 0.01 mg-GAE g⁻¹ and 24.32 ± 1.06 mg-CE g⁻¹, respectively) under the conditions of 30 °C, 70% moisture, 1 × 10⁶ spores g⁻¹, pH 5.5, and a concentration of MgSO₄ of 0.76 g L⁻¹. Based on the Box–Behnken experimental design and the subsequent quantification of hydrolyzable and condensed polyphenols, treatment 8 was selected as optimal. This selection was predicated on treatment 8, having demonstrated the highest accumulation of hydrolyzable and condensed tannins while exhibiting no statistically significant difference in the accumulation of condensed tannins compared to other treatments.

The selection of pH and MgSO₄ concentration as independent variables for NDGA accumulation was based on a previous study conducted by Coronado-Contreras (2020) [38] within our research group. That study evaluated NDGA production by SSF using creosote bush leaves and employed a Plackett-Burman experimental design to identify factors influencing NDGA accumulation. The factors evaluated were temperature, pH, humidity, inoculum (spores g⁻¹), and the concentrations of NaNO₃ (g L⁻¹), MgSO₄ (g L⁻¹), and KCl (g L⁻¹). The results indicated that only the concentration of MgSO₄ and pH had a significant influence on NDGA accumulation. Therefore, the present study focused on evaluating only the concentration of MgSO₄ and pH as independent variables to optimize the process of using a Box–Behnken experimental design. The other factors (NaNO₃ (6 g L⁻¹), KCl (0.26 g L⁻¹), temperature (30 °C), humidity (70%), and inoculum (1x10⁶ spores g⁻¹) were kept constant based on the previous findings.

The Pareto chart (Figure 4) represented the relative influence of pH and MgSO₄ concentration (g L⁻¹) on the response variable (condensed tannins), determined by the Box–Behnken experimental design. The pH was the only factor that exceeded the 95% confidence threshold, signifying a significant positive linear effect. This observation was corroborated by a statistically significant linear correlation between pH and condensed tannin content. Specifically, the data demonstrated a direct and proportional relationship: increases or decreases in pH can lead to predictable changes in condensed tannin content.

3.3. HPLC-MS Analysis of Compounds in Fermented Extracts of Creosote Bush Leaves

Once we established the best fermentation treatment of SSF according to the Box–Behnken experimental design (treatment 8), the compounds present in the fermentation extracts were characterized by HPLC-MS. The identified compounds are shown in

Table 3. Compounds present in fermentation extracts of creosote bush leaves.

Retention time (min)	Mass [M-H]−	Compound	Family
22.451	304.9	(+)-Gallocatechin	Catechins
25.562	283.9	Methylgalangin	Methoxyflavonols
27.071	288.9	(+)-Catechin	Catechins
29.740	754.7	Quercetin 3-O-rhamnosyl-rhamnosyl-glucoside	Flavonols
31.425	305.0	(−)-Epigallocatechin	Catechins
33.215	608.9	Quercetin 3-O-rutinoside	Flavonols
35.462	596.9	Delphinidin 3-O-sambubioside	Anthocyanins
38.108	333.0	Gallic acid 4-O-glucoside	Hydroxybenzoic acids
39.529	386.9	Medioresinol	Lignans
43.635	300.9	Ellagic acid (EA)	Hydroxybenzoic acids
47.439	330	3,7-Dimethylquercetin	Methoxyflavonols
49.317	298.9	4-Hydroxybenzoic acid 4-O-glucoside	Hydroxybenzoic acids
51.841	347.0	5-Heptadecylresorcinol	Alkylphenols
54.047	301.1	Nordihydroguaiaretic acid (NDGA)	Lignans
56.551	313.0	Cirsimaritin	Methoxyflavones
58.322	630.7	Pelargonidin 3,5-O-diglucoside	Anthocyanins

.

According to the identification via HPLC-ESI-MS, the presence of EA and NDGA was demonstrated, with NDGA being the major compound. Both compounds were quantified, obtaining concentrations of 1.20 ± 0.32 mg g⁻¹ for EA and 7.39 ± 0.52 mg g⁻¹ of NDGA.

3.4. Antioxidant Activity of Fermentation Extracts from Creosote Bush Leaves

The fermentation extracts recovered from SSF of creosote bush leaves demonstrated antioxidant activity by the two assays evaluated, DPPH inhibition was 55.69 ± 1.51%, and the ABTS assay had the highest inhibition percentage value of 84.84 ± 1.47% (

Table 4. Antioxidant activities of best treatment of SSF.

Antioxidant Assay	Inhibition (%)
DPPH	55.69 ± 1.51
ABTS	84.84 ± 1.47

).

4. Discussion

In the SSF process, water plays a key role. The WAC value determines the ability of the evaluated material to absorb and retain water, as water has a significant effect on the SSF process. Therefore, the WAC value is considered important and should be considered [39]. Substrates subjected to SSF usually exhibit a higher water absorption capacity (WAC) [40], which is desirable in SSF processes, since the substrate can lose moisture [41].

Creosote bush leaves can withstand a maximum moisture value of 73.76% (Table 2) before free water appears in the medium, which is not suitable in SSF because high moisture percentages contribute to particle agglomeration and interfere with gas exchange processes and may promote bacterial growth [42]. Furthermore, in SSF processes, filamentous fungi are employed, hence the common practice of operating within a moisture value of 30–85%. Nonetheless, the substrate utilized must possess a moisture value range of 20–70% to facilitate SSF with filamentous fungi [43]. According to the factors mentioned above, it was decided to conduct the fermentation at 70% moisture value, which is a value widely used in SSF with filamentous fungi. Buenrostro-Figueroa et al. (2023) [44] obtained comparable values for the evaluated parameters when assessing pomegranate by-products. They reported a moisture content of 11.86% and a WAC value of 4.38 g-gel g⁻¹ dry material. Larios-Cruz et al. (2019) [45] reported that grape by-products exhibited similar values; they reported a moisture content of 3.65%, a solids content of 96.35%, and a WAC value of 4.30 g-gel g⁻¹ dry material. Consequently, it is demonstrated that the evaluated values of the creosote bush are within the values obtained from other supports previously evaluated in the SSF process, and thus, it can be employed as support to conduct SSF.

Proximal chemical analysis (Table 2) revealed a fat content of 5.11 ± 0.08%, suggesting the presence of lipids crucial for energy transport and storage, particularly relevant for adaptation in arid environments [46]. Fiber content was 16.82 ± 3.47%, due to the lignocellulosic structure of the leaves. Protein content, measured via the Kjeldahl method, was 1.28 ± 0.01%, indicating nitrogen availability, potentially serving as a nitrogen source for A. niger GH1. Total sugars were 37.06 ± 0.09%, encompassing structural and energy-providing carbohydrates [46]. Ash content, reflecting inorganic mineral composition, was 9.41 ± 0.87%, influenced by soil mineral content. The proximal characterization of creosote bush suggests that the SSF process can be conducted using the creosote bush leaves as support/substrate.

Box–Behnken design treatments significantly altered tannin release compared to controls, suggesting that the SSF process did influence the release of hydrolyzable and condensed tannins (Figure 3). Specifically, all treatments (1–9) exhibited elevated levels of hydrolyzable tannins, with treatment 8 demonstrating the highest accumulation of hydrolyzable tannins (36.17 ± 0.01 mg-GAE g⁻¹). Similarly, many treatments, except for treatment 3, showed increased condensed tannin levels compared to controls. Notably, treatments 9 and 8 yielded the highest condensed tannin concentrations, measuring 27.07 ± 0.70 mg-CE g⁻¹ and 24.32 ± 1.06 mg-CE g⁻¹, respectively.

The complex cell wall matrix of plant biomass, primarily composed of structural polysaccharides (cellulose, pectin) and proteins, can associate with various phenolic compounds, including tannins, phenolic acids, flavonoids, and lignans [47]. These interactions are known to impede the efficient extraction and release of such compounds. To address this limitation, the application of microbial enzymes, particularly those derived from filamentous fungi, has been explored to facilitate cell wall degradation and enhance the liberation of cell wall-bound compounds [48,49]. The genus Aspergillus spp. is widely employed in this context due to its capacity to produce a diverse array of hydrolytic enzymes capable of degrading various lignocellulosic cell wall components, such as cellulases and xylanases, among others [50]. Specifically, A. niger GH1 has been reported to produce these hydrolytic enzymes and, notably, tannin-degrading enzymes like ellagitannase and tannase, which are considered key enzymes in the release of phenolic compounds of interest, such as ellagic acid and other phenolic compounds [51].

The observed variations in tannin accumulation across the different Box–Behnken design treatments highlight the sensitivity of tannin release to the manipulated variables. treatment 8, characterized by a pH of 5.5, resulted in the highest levels of both hydrolyzable and elevated condensed tannins (treatment 8 condensed tannins were statistically similar to treatment 9). Conversely, treatment 3, conducted at a pH of 4.5, displayed minimal condensed tannin accumulation. This observation suggests a potential positive correlation between the pH of the fermentation medium and the release of condensed tannins (as visually represented in Figure 4). This aligns with the reported microbial resistance of condensed tannins [52], where higher pH may facilitate their release. Consequently, high pH conditions may favor the release of condensed tannins.

Coronado-Contreras (2020) [38], in a prior study conducted in our group, established optimal conditions for NDGA production via SSF employing A. niger GH1 and utilizing creosote bush leaves as support. This study identified pH and MgSO₄ concentration as key factors influencing NDGA accumulation (6–12 mg g⁻¹ NDGA) through Plackett-Burman experimental design. The present study employed a Box–Behnken design with the same variables, with the objective of process optimization, and achieved a maximum of 7.39 mg g⁻¹ NDGA. While optimal conditions were not attained, the evaluation of these factors, despite not yielding a substantial increase in NDGA accumulation, contributed to the observed accumulation. This suggests that while the selected variables are influential, the specific conditions and experimental design significantly impact the final NDGA yield.

This difference in evaluated factors in both studies, as well as the experimental designs, could explain the difference in NDGA accumulation values observed between the studies. The variation in experimental designs and evaluated factors underscores the complexity of NDGA production through SSF using creosote bush leaves and the need for a detailed characterization of each factor’s influence.

The HPLC-MS analysis of the SSF extracts revealed NDGA as the predominant compound, quantified at 7.39 mg g⁻¹, EA was also found to be present at 1.20 mg g⁻¹ (Table 3). These findings suggest that SSF significantly modulated the release and accumulation of these bioactive compounds. This idea is supported by the observation that several compounds identified in the fermentation extracts were not previously reported in studies employing alternative extraction methodologies. Specifically, Aguirre-Joya et al. (2018) [19] reported kaempferol as the major compound, followed by NDGA and quercetin, in aqueous extracts of creosote bush leaves analyzed by HPLC-MS. While NDGA and quercetin were also identified in the present study, kaempferol was notably absent from the SSF extracts. Furthermore, the detection of EA in the fermentation extracts reinforces the notion that SSF significantly altered the phytochemical profile. García et al. (2018) [53] investigated the anthelmintic activity of hydro-methanolic extracts of creosote bush, reporting the presence of sesamin, gallocatechin, peonidin 3-O-rutinoside, methylgalangin, epigallocatechin 7-O-glucuronide, and epigallocatechin via HPLC-MS analysis. Notably, sesamin, peonidin 3-O-rutinoside, and epigallocatechin 7-O-glucuronide, identified by García et al. (2018) [53], were not detected in the present SSF extracts (Table 3). This disparity highlights the significant influence of extraction technique and solvent selection on the recovery of phenolic compounds from creosote bush.

The DPPH assay revealed that the fermentation extract of treatment 8 demonstrated a significant radical scavenging activity, achieving a percentage inhibition of 55.69% (Table 4). The DPPH assay quantifies antioxidant capacity by measuring the reduction in the stable DPPH radical, a process driven by the transfer of a hydrogen atom to the radical’s nitrogen atom [54]. This transfer is facilitated by antioxidants capable of donating hydrogen, particularly from hydroxyl groups. In the present study, HPLC-MS analysis of treatment 8 identified the presence of NDGA and EA, compounds known for their potent antioxidant properties and the presence of readily available hydroxyl groups. Consequently, the observed DPPH radical scavenging activity in treatment 8 is likely attributable to the hydrogen-donating capabilities of NDGA and AE, among other compounds. The ABTS assay is similar to the DPPH assay; by this method, it is possible to examine the antioxidant activity of hydrophilic and lipophilic compounds [55]. The ABTS inhibition value obtained in this study (84.84%) shows that almost all the free radicals present in the reaction were inhibited. This was similar to the results obtained by Aguirre-Joya et al. (2018) [19], where a 97% inhibition was obtained using polyphenolic compounds from the creosote bush. It is worth mentioning that due to the presence of different compounds in the plant extracts, these directly influence the antioxidant capacity. Furthermore, it should be noted that for practical purposes in this study, the fermentation extracts were diluted (1:50 v/v) to conduct the antioxidant techniques appropriately, and perhaps this influenced the antioxidant activity.

5. Conclusions

Creosote bush leaves proved to be viable for use as a support/substrate to perform a SSF due to physical tests demonstrating that they possess a WAC value of 3.62 ± 0.02 and support a maximum humidity above 70%, the micro-organism A. niger GH1 was able to grow and invade the plant material. Although it was not possible to optimize the SSF process, the evaluated conditions allowed the extraction and characterization of a total of sixteen different compounds, highlighting the presence of NDGA and EA, which were quantified in treatment 8. The fermentation extracts of treatment 8 also exhibited significant in vitro antioxidant activity, with inhibition percentages of 55.69% in the DPPH assay and 84.84% in the ABTS assay. This demonstrates that the compounds present in the extracts possess biological activities that can be beneficial and used in various industries. To achieve optimal yields and further enhance the production of these valuable compounds, future research should focus on elucidating the specific parameters required for optimizing the SSF process using creosote bush leaves.