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Article

Solid-State Fermentation with Rhizopus oryzae: Enhancing Antioxidant and Phenolic Content in Pigmented Corn

by
Ulises Ramírez-Esparza
1,
Andrés J. Ordoñez-Cano
1,
Leticia X. López-Martínez
2,
José C. Espinoza-Hicks
3,
Mónica Alvarado-González
4,
Juan A. Ascacio-Valdés
5 and
José Juan Buenrostro-Figueroa
1,*
1
Biotechnology and Bioengineering Laboratory, Research Center in Food and Development, Delicias 33089, Chihuahua, Mexico
2
SECIHTI, Research Center in Food and Development, Hermosillo 83301, Sonora, Mexico
3
School of Chemistry, Universidad Autónoma de Chihuahua, Chihuahua 31125, Chihuahua, Mexico
4
Microbiology and Molecular Biology Laboratory, Research Center in Food and Development, Delicias 33089, Chihuahua, Mexico
5
Bioprocesses & Bioproducts Research Group, Food Research Department, School of Chemistry, Universidad Autónoma de Coahuila, Saltillo 25280, Coahuila, Mexico
*
Author to whom correspondence should be addressed.
Resources 2025, 14(10), 158; https://doi.org/10.3390/resources14100158
Submission received: 11 August 2025 / Revised: 29 September 2025 / Accepted: 1 October 2025 / Published: 9 October 2025
(This article belongs to the Special Issue Resource Extraction from Agricultural Products/Waste: 2nd Edition)
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Abstract

Corn is one of the most widely cultivated cereal crops and is rich in antioxidant compounds, especially phenolics. However, many of these are bound to cell wall components, requiring pre-treatment for release. Solid-state fermentation (SSF) with Rhizopus oryzae has been used to enhance antioxidant capacity in grains and legumes, though its application in pigmented corn (PC) has not been reported. This study evaluated R. oryzae growth on PC via SSF and its effect on phenolic compound release and antioxidant capacity (AC). Variables such as temperature, pH, inoculum, and medium salts were tested for their influence on phenolic release and AC. Nutrient changes in PC due to SSF were also examined. HPLC-MS was used to analyze the phenolic compounds’ profile. R. oryzae grew effectively on PC, increasing total phenolic content (TPC) and AC by 131 and 50%, respectively. The pH was found to negatively impact phenolic release. The SSF also raised protein content by 10% and reduced carbohydrates and fiber by 3 and 8%. Thirteen phenolic compounds were identified, including Feruloyl tartaric acid ester and p-Coumaroyl tartaric acid glycosidic ester, with known anti-inflammatory properties. This process offers a sustainable method for enhancing the functional properties of pigmented corn.

1. Introduction

Due to its nutritional and phytochemical profile, corn (Zea mays L.) is one of the most important crops in the world [1]. A diversity of phenolic compounds has been identified in their composition, mainly phenolic acids such as ferulic, p-coumaric, caffeic, and anthocyanins in pigmented corn [2,3]. These compounds exhibit antioxidant, anti-inflammatory, and antimutagenic properties and potential beneficial effects on human health [4,5]. However, many of these compounds are found in conjugated forms or covalently bound to the plant matrix, mainly cell walls, limiting their bioavailability and physiological and functional use in food products [5,6]. These compounds can be released using conventional techniques, such as maceration, or emerging techniques, including ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), and biotechnological methods [7].
In this context, solid-state fermentation (SSF) has positioned itself as a cheap and effective biotechnological alternative process for valorizing plant matrices rich in phenolic compounds, such as corn. Through the enzymatic action of filamentous fungi, particularly species of the genus Rhizopus, it is possible to induce structural modifications in the substrate that favor the release, transformation, and in some cases, the synthesis of new bioactive metabolites previously inaccessible [8,9]. Within the Rhizopus genus is R. oryzae, which has interesting characteristics for its application in food. For example, it grows at room temperature (25 to 35 °C) and produces enzymes such as amylases, proteases, and lipases during its growth, which can help increase the bioavailability of nutrients by performing hydrolysis on the cell wall of plant material [10]. Several foods use Rhizopus to obtain textures and flavors. In Asia, it is very common to use this fungus in the production of various foods such as tempeh, which is a fermented grain, legume, or fruit product made with the fungus R. oryzae [11].
This fermentation process also has advantages compared to other processes, such as liquid fermentation, including low water consumption, high energy efficiency, and low contamination, making it a sustainable option for developing functional ingredients [12].
However, the success of this process largely depends on the careful selection and management of critical parameters, such as moisture, temperature, pH, substrate type, particle size, carbon/nitrogen ratio, inoculum, and fermentation time. These parameters directly influence microbial activity and enzyme expression, which in turn affect the profile and quantity of phenolic compounds released or transformed [13,14,15]. Understanding and optimizing these parameters not only maximizes the production of bioactive compounds but also provides the basis for the design of functional foods with specific properties.
Functional foods have become increasingly important due to the rising prevalence of chronic non-communicable diseases and consumer interest in adopting healthier and more preventive diets [4]. In addition to providing essential nutrients, these products offer specific physiological benefits, such as modulation of the gut microbiota, enhancement of the immune response or reduction in oxidative stress [16,17]. In this sense, the development of functional foods from traditional raw materials such as corn, through sustainable biotechnological processes such as SSF, represents a strategic opportunity for food innovation and improvement of public health. For this purpose, this study aims to evaluate the use of SSF with R. oryzae to improve the content, availability, and profile of phenolic compounds in PC. It also considers the influence of key process parameters in producing a fermented food product with functional potential for food applications.

2. Materials and Methods

2.1. Raw Material

Grains of pigmented corn (PC) harvested in the Sierra Tarahumara region (Madera, Chihuahua; coordinates: 29.243298, −108.113481) between October and November 2021 were selected for this study. After collection, samples were placed in black bags and transported to the Biotechnology and Bioengineering Laboratory at CIAD Delicias. There, they were disinfected using UV radiation for 30 min and subsequently stored in the dark until further use.

2.2. Physicochemical Characterization of Blue Corn

The contents of fats (AOAC 933.05), proteins (AOAC 960.521), crude fiber, (AOAC 985.29) moisture (AOAC 930.15), carbohydrates (by difference) and ash (AOAC 900.02) were analyzed following the standardized methods outlined by the Association of Official Agricultural Chemists (AOAC) [18].

2.3. Microorganism

For this study, Rhizopus oryzae from the fungal culture collection of the Bioprocesses & Bioproducts Research Group, Food Research Department at FCQ-UadeC was utilized. The strain was preserved at −20 °C in a glycerol and skimmed milk solution (1:9 v/v). The spores were cultured on potato dextrose agar (PDA-Bioxon) and incubated at 30 °C for seven days. Subsequently, the reactivated spores were transferred to 250 mL Erlenmeyer flasks containing 30 mL of PDA and incubated at 30 °C for seven days. The spores were collected using a sterile 0.01% (v/v) Tween-80 solution and counted using a Neubauer chamber.

2.4. Effect of Different Factors on the Release of TPC

Whole grains were soaked following the methodology described by Chen et al. [5] with modifications to achieve a moisture content of 35% through a 2 h soaking period. Corn grains were used as the substrate for fermentation (15 g per tray reactor). A 2k Box-Hunter & Hunter (BHH) factorial design was used to evaluate the effect of temperature, pH, inoculum concentration and salts in the medium, as shown in Table 1. The extracts were obtained after 60 h of SSF. To obtain extracts, 3 g of fermented corn were milled (≤1 mm, Hamilton Beach, Ontario, Canada) and treated with 10 mL of methanol-water-lactic acid (80:19:1 v/v/v). The samples were vortexed, filtered through 25 μm filter paper (Whatman), transferred into 2 mL vials, and stored at −18 °C until analysis.
The extracts were obtained after 60 h of SSF with R. oryzae and used to determine the TPC and AC assays.

2.5. Effect of SSF on the Physicochemical Composition of Blue Corn

In this stage, another SSF (300 g) was carried out in a tray reactor (1183 cm3) using PC as substrate with the best process conditions previously selected in Section 2.4 (40 °C, pH 5, 1 × 107 sp/g, 1 g/L KH2PO4, 2 g/L MgSO4·7H2O and 4 g/L (NH4)2SO4). The fermented grains were dried and milled to fine particles (≤1 mm) that were used to perform the proximal analysis [18] and to obtain the phenolic compound profile by HPLC-MS.
The results obtained were compared to determine the effect of SSF on the physicochemical composition, TPC and AC of pigmented corn.

2.6. Analytical Analysis

2.6.1. Determination of Phenolic Content

The hydrolysable phenolic content was quantified using the Folin–Ciocalteu method according to Wong-Paz et al. [19]. For that, 20 μL of extract were mixed with 20 μL of Folin–Ciocalteu reagent in a microplate. After a 5 min incubation, 20 μL of 0.01 M sodium carbonate were added, and let to stand for 5 min. Subsequently, 125 μL of distilled water were added followed by a final agitation, the absorbance was measured at 790 nm using a MULTISKAN GO microplate reader (Thermo Fisher Scientific, Vantaa, Finland). Gallic acid standard was used in the range of 0 to 200 mg/L. Results were expressed as milligrams of gallic acid equivalent per gram of dry matter (mg GAE/g dm).
For condensed phenolic content, extracts were evaluated as described Hernández et al. [20] adapted to microplate. The sample (250 μL) was mixed in screw-cap tubes with 1.5 mL of HCl–n-butanol solution (1:9 v/v) and 50 μL of ferric reagent. The tubes were capped and heated in a boiling water bath at 100 °C for 40 min, and after cooling to room temperature, 200 μL of sample was transferred to a microplate, and absorbance was measured at 460 nm using a MULTISKAN GO microplate reader. Results were expressed as catechin equivalents per gram of dry matter (mg CE/g dm) using a calibration curve of 0–1000 mg/L.
Total phenolic content (TPC) was calculated as the sum of hydrolysable and condensed phenolic contents and reported as milligrams per gram of dry mass (mg/g dm).

2.6.2. Determination of Antioxidant Capacity

ABTS Antioxidant Assay
The ABTS+ radical reagent was generated by mixing 2.45 mL of 7 mM ABTS (2,2-0-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) with 12.5 mL of 2.45 mM potassium persulfate (K2S2O8) and incubated in the dark for 16 h to generate the ABTS+ radical Torres-León et al. [21]. The absorbance of ABTS+ solution was adjusted at 0.7 ± 0.2 at 734 nm with ethanol. For the assay, 10 μL of sample was mixed with 190 μL of ABTS+ solution in a microplate, and absorbance was measured after 1 min in a microplate reader. Results were expressed as Trolox equivalents per gram of dry matter (mg TE/g dm) using a calibration curve of 0–200 mg/L.
DPPH Antioxidant Assay
The 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay [22] was adapted to microplate. For that, 7 μL of sample were mixed with 193 μL of DPPH solution (60 μM in methanol) and left to stand. After 30 min, the absorbance was measured at 517 nm in a microplate reader. Results were expressed as milligrams of Trolox equivalents per gram of dry matter (mg TE/g dm) using a calibration curve of 0–200 mg/L.
Ferric Reducing Antioxidant Power (FRAP) Assay
The FRAP assay was determined according to López-Cárdenas et al. [23]. The FRAP reagent was prepared by combining 50 mL of a 0.3 M acetate buffer solution (pH 3.6), 5 mL of a 10 mM TPTZ (2,4,6-tri(2-pyridyl)-s-triazine) solution, and 5 mL of a ferric chloride solution. The mixture was then incubated at 37 °C for 30 min. For the assay, 18 μL of distilled water, 6 μL of the extract, and 180 μL of the prepared FRAP reagent were mixed and incubated at 37 °C for 60 min. Absorbance was measured at 595 nm using a microplate reader (Thermo Fisher Scientific, Vantaa, Finland). Results were expressed as milligrams of Fe2+ per gram of dry matter (mg Fe2+/g dm) using a calibration curve of iron sulfate of 0–800 mg/L.

2.6.3. Identification of Phenolic Compounds by HPLC-MS

The phenolic profile was determined using a reversed-phase high-performance liquid chromatography on a Varian HPLC system, including an autosampler (Varian ProStar 410, Palo Alto, CA, USA), a ternary pump (Varian ProStar 230I, Palo Alto, CA, USA), and a PDA detector (Varian ProStar 330, Palo Alto, CA, USA). The sample (5 µL) was injected and separated using a Denali C18 column (150 mm × 2.1 mm, 3 µm, Palo Alto, CA, USA) at 30 °C. Methanol was used as wash phase, and formic acid (0.2% v/v; solvent A) and acetonitrile (solvent B) as eluents. The gradient conditions were as follows: 3% B and 97% A from 0 to 5 min, 9% B and 91% A from 5 to 15 min, 16% B and 84% A from 15 to 45 min, and 50% B and 50% A thereafter, the flow rate was maintained at 0.2 mL/min and elution was monitored at 245, 280, 320 and 550 nm. Mass spectrometric analysis was performed using a Varian 500-MS IT Mass Spectrometer (Palo Alto, CA, USA) equipped with an electrospray ion source, operating in negative mode at a temperature of 350 °C, capillary voltage of 90.0 V, and spray voltage of 5.0 kV. Data processing was carried out using MS Workstation software (V 6.9). Mass spectra were recorded in a m/z range of 50–2000 [24].

2.7. Statistical Analysis

A 2k BHH design was set up to identify the factors significantly affecting the TPC extraction. For this purpose, six factors (inoculum size, temperature, pH, MgSO4, (NH4)2SO4, and KH2PO4) at two levels each were used to construct a condensed matrix with six 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 LSD 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 TPC and AC (ABTS, DPPH, and FRAP) were determined using Pearson’s correlation coefficient (p < 0.01).

3. Results

3.1. Effect of Different Factors on the Release of TPC

The effect of various factors of the SSF process (temperature, pH, inoculum size, and salt concentration (KH2PO4, MgSO4, (NH4)2SO4)) was evaluated using a 2k BHH experimental design on corn pigmented TPC with R. oryzae.
Figure 1 shows the Pareto diagram that categorizes the effect of each factor evaluated on the response variable, with the one that exceeds the dotted center line being significant.
Table 2 summarizes the effects of various evaluated factors on the response variables (TPC and AC), as indicated by the Pareto diagrams. Notably, pH emerged as the factor with a significant impact on all response variables, showing a negative correlation with each. This means that as the initial pH of the medium increases, the values of the response variables decrease. This behavior can be explained by the fact that each microorganism has an optimum pH value for growth, and deviations from this optimum value can directly influence the microbial development in the corn grain [25]. These changes in pH can also affect the activity and stability of the enzymes produced by the fungus during its metabolism, because they may lose their catalytic activity due to the structural alterations of the enzymes as a result of the pH change [26,27].
On the other hand, the inoculum size showed a positive correlation, indicating that an increase in inoculum concentration favors the release of TPC. This finding aligns with the reported by Egbune et al. [28], who observed an increase in free TPC with larger inoculum sizes using millet as a substrate for R. oligosporus. Additionally, KH2PO4 also showed a positive correlation, indicating that increasing its concentration enhances AC against the ABTS radical. MgSO4·7H2O exhibited a similar effect, but specifically against the DPPH radical. Fan et al. [29] reported a similar trend, noting that higher concentrations of K+ and Mg2+ ions in various substrates result in increased AC against the ABTS and DPPH radicals, respectively.
Ammonium sulphate showed a negative correlation with the AC determined by the FRAP assay. This may be attributed to the ability of R. oryzae to produce siderophores, which are compounds that chelate iron [30]. The presence of these chelating agents might interfere with the assessment of AC using the FRAP assay [31]. It is important to highlight that there are no specific references emphasizing the direct impact of these salts on the AC methodologies evaluated here. Consequently, they may be overestimated due to potential interferences in the determination of AC. For instance, phenolic compounds such as anthocyanins, caffeic acid and ferulic acid, which have been identified in corn, react slowly with the FRAP reagent [31]. Therefore, the reaction time used in the methodology might have been too short to accurately reflect the true antioxidant potential of the compounds present in the fermented extracts.
One disadvantage of the DPPH assay is that it can produce overestimated results because some compounds absorb at the same wavelength. Also, this assay utilizes an organic solvent, which restricts the measurement of AC for water-soluble compounds. In contrast, the ABTS method can effectively assess the AC of hydrophilic compounds [31,32].
Table 3 presents the results for TPC for each treatment. The highest release was achieved in treatment 6 (5.549 mg/g dm), which is 1.15-fold greater than the lowest value observed in treatment 3 and 1.31-fold compared to the unfermented control (2.4 ± 0.03 mg/g dm). These changes in phenolic compound concentrations can be attributed to various causes, e.g., the action of enzymes (cellulases, proteases, lipases) produced by R. oryzae during its development, which help to release phenolic compounds bound to cell wall components through enzymatic degradation [33,34]. In turn, the change in concentrations may be influenced by abiotic parameters, such as temperature, which directly affects microbial growth by accelerating or inhibiting enzymatic reactions and thus interrupting microbial growth [35]. Minerals (metal ions) available during the development of the microorganism also play an important role, as many of them participate in a direct way as cofactors of enzymes, such as magnesium, potassium and phosphorus, which greatly affect the development of the microorganism [36].
Based on the obtained results, the best conditions for a better release of TPC from pigmented corn fermented with R. oryzae were temperature 40 °C, pH 5, inoculum size 1 × 107 sp/g, 1 g/L KH2PO4, 2 g/L MgSO4·7H2O and 4 g/L (NH4)2SO4.

3.2. Effect of SSF on the Physicochemical Composition of Blue Corn

The effect of SSF on the chemical composition and AC of pigmented corn, using R. oryzae, was evaluated using the best treatment (treatment 6). Figure 2 shows the growth of R. oryzae on pigmented corn grain after 60 h of culture under conditions that promote higher TPC release with AC, as observed in treatment 6. The below images in the figure provide a more detailed view of mycelium development during the fermentation process. The results obtained for the physicochemical composition TPC and AC are shown in Table 4.
When comparing the fermented material with the control, a 10% increase in protein content was observed. This finding aligns with the reported by Sukma et al. [37], who fermented rice with R. oryzae. In another study, rice was used as a substrate in SSF with R. oryzae, Kupski et al. [38] reported a 37.6% increase in protein compared to the control. This increase in protein content can be attributed to the microorganism utilized the substrate as a source of carbon and energy during SSF, which promotes its grow and, consequently, enhances fungal protein content [39].
The total fat content after the SSF process increased by 1.86 times compared to unfermented corn. This increase may be due to the growth of R. oryzae, which use available nutrients to synthesize lipids that become part of the mycelial structure [40]. Moreover, R. oryzae has been reported to produce lipases, enzymes that break down lipids bound to other corn components, such as sugars or proteins [10,41]. In terms of fiber content, a decrease of 8% was observed, similar to the 5% reduction reported by Sukma et al. [42] in their study on banana flour fermented with R. oryzae. Similarly, Olugosi et al. [43] reported a 5% decrease in fiber content in cocoa husk flour fermented with R. stolonifer. These declines in fiber are attributed to the ability of R. oryzae to produce different cell wall degrading enzymes, including cellulases, hemicellulases and proteases [10].
A 3% decrease in carbohydrate content was observed, similar to the reported by Sukma et al. [42] on banana flour fermented with R. oryzae. During its growth and development, R. oryzae produces enzymes that break down polysaccharides into simpler compounds, such as glucose. The fungus then uses glucose as a carbon source for its biomass development, resulting in a reduction in carbohydrate content [42,44].
When comparing the TPC, an increase of 131% was observed with respect to the unfermented grain, a value similar to 125% increase reported by Chen et al. [5] in the phenolic content of corn fermented with Monaskus anka. Buenrostro-Figueroa et al. [35] achieved a remarkable 439% increase in the phenolic content of figs fermented with R. oryzae under optimal conditions of process.
On the other hand, an increase in AC of up to 60% was observed in fermented corn compared to unfermented control. This positive increase in AC is consistent with findings from several researchers. For instance, Buenrostro-Figueroa et al. [45] reported an enhancement of up to 481% in AC through an optimized process that involved fermenting pomegranate peel with A. niger. Janarny and Gunathilake [46] achieved a 44% increase in AC using different types of rice fermented with R. oryzae. Dulf et al. [47] demonstrated a 35% increase in AC by fermenting plums with two fungal strains, A. niger and R. oligosporus. Table 5 presents the 11 compounds found in the SSF of pigmented corn.
After fermentation, some phenolic compounds were only found in the fermented corn, suggesting that these compounds may have been released through the enzymatic action associated with the fermentation process involving R. oryzae.
Some of these phenolic compounds are of great interest in the industrial and food sectors due to their remarkable biological activities [5,21]. Caffeic acid and its derivatives are present in various cereals and plants. Several studies have highlighted the potential health benefits of this compound, which include anti-inflammatory, antioxidant, neuroprotective, and anticancer effects [48,49,50]. Quercetin 3-O-acetyl-rhamnoside is an acetylated derivative of Quercetin found in fruits such as blackberries, grapes and raspberries. This compound offers various health benefits, including the ability to inhibit α-glucosidase, which helps reduce hyperglycemia in individuals with diabetes [51]. Psoralen is another phenolic compound found in various vegetables and plants, such as figs and celery. It is medically significant due to its applications in treating conditions like osteoporosis, inflammation and cancer [52]. Ferulic acid, the most abundant phenolic acid in corn, can form a conjugated phenolic acid known as feruloyl tartaric acid when combined with a tartaric acid [53]. This compound has beneficial qualities for the body, including antioxidant and anti-inflammatory properties, making it valuable in the production of functional foods [54].
Enzyme activity during microbial development can vary based on growth conditions and the availability of nutrients in the substrate. These enzymes can either release compounds from the blue corn cell wall or transform existing compounds into new ones. Such fluctuations may explain the appearance or disappearance of certain compounds observed during fermentation (Table 5). Another factor contributing to this variability is the influence of the physicochemical conditions of the medium (such as pH, temperature, and oxygen availability) which can affect compound stability and transformation. For instance, changes in pH can alter the chemical structure of bioactive compounds. Furthermore, the continuous enzymatic degradation of the blue corn cell wall by fungi may lead to the release of previously bound compounds.
These results demonstrate the efficiency of SSF as an alternative method for enhancing the content of TPC and AC in plant matrices. The data show a positive correlation between TPC and AC, indicating that as phenolic compounds bound to the constituents of the plant matrix are released during SSF, both TPC and AC increase. This increase is a consequence of the degradation of the cell wall components in pigmented corn, where many phenolic compounds are located, facilitated by the enzymatic activity of R. oryzae on the corn kernels [55].

4. Conclusions

The results of this study suggest that the chemical composition of pigmented corn makes it a suitable candidate for use as a substrate in SSF processes. The fungus R. oryzae was able to grow using pigmented corn grain as both the substrate and sole nutrient source. Among the process parameters evaluated, only pH had an impact on TPC and AC. The best conditions for SSF of pigmented corn with R. oryzae were temperature 40 °C, pH 5, inoculum size 1 × 107 sp/g, 1 g/L KH2PO4, 2 g/L MgSO4·7H2O and 4 g/L (NH4)2SO4. Under these conditions, SSF of corn pigmented with R. oryzae led to a 10% increase in protein content, while reducing carbohydrate and fiber content by 8 and 3%, respectively. Furthermore, the SSF using R. oryzae resulted in a 131% increase in the extraction of free TPC, and the AC of the fermented extracts improved on average by up to 50%.
This proposed bioprocess enhances the physicochemical and functional characteristics of the pigmented corn. With its high AC and improved chemical composition, the processed grain could serve as an excellent material for developing functional foods.

Author Contributions

Conceptualization, J.J.B.-F., J.A.A.-V. and J.C.E.-H.; Methodology, U.R.-E. and A.J.O.-C.; software, U.R.-E. and J.A.A.-V.; Validation, M.A.-G., L.X.L.-M. and J.C.E.-H.; Formal analysis, U.R.-E. and A.J.O.-C.; Investigation, U.R.-E. and J.J.B.-F.; Resources, J.J.B.-F.; data curation, U.R.-E. and J.A.A.-V.; Writing—original draft preparation, U.R.-E.; writing—review and editing, all authors; supervision, M.A.-G., L.X.L.-M., J.A.A.-V., J.C.E.-H. and J.J.B.-F.; project administration, J.J.B.-F.; funding acquisition, J.J.B.-F. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

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

Acknowledgments

U.R.-E. thanks SECIHTI for the postgraduate scholarship awarded for his doctoral studies.

Conflicts of Interest

The authors declare no conflicts of interest.

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Resources 14 00158 g001
Figure 1. Pareto diagram for TPC.
Figure 1. Pareto diagram for TPC.
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Figure 2. Growth of R. oryzae on pigmented corn: unfermented control (A,C) and fermented at 60 h with treatment 6 (B,D).
Figure 2. Growth of R. oryzae on pigmented corn: unfermented control (A,C) and fermented at 60 h with treatment 6 (B,D).
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Table 1. BHH matrix used to evaluate the effect of 6 factors (A, B, C, D, E and F) on free TPC of PC with R. oryzae at SSF.
Table 1. BHH matrix used to evaluate the effect of 6 factors (A, B, C, D, E and F) on free TPC of PC with R. oryzae at SSF.
TreatmentABCDEF
1−1−1−1111
21−1−1−1−11
3−11−1−11−1
411−11−1−1
5−1−111−1−1
61−11−11−1
7−111−1−11
8111111
CodeFactorLow level (−1)High level (1)
ATemperature (°C)3040
BpH56
CInoculum (sp/g)1 × 1061 × 107
DKH2PO4 (g/L)12
EMgSO4·7H2O (g/L)12
F(NH4)2SO4 (g/L)48
Table 2. Effect of processing conditions on TPC and AC of PC.
Table 2. Effect of processing conditions on TPC and AC of PC.
FactorTPCABTSDPPHFRAP
Temperature (°C)NSNSNSNS
pH−−−−
Inoculum (sp/g)+NSNSNS
KH2PO4 (g/L)NS+NSNS
MgSO4·7H2O (g/L)NSNS+NS
(NH4)2SO4 (g/L)NSNSNS−
+: Positive effect, −: Negative effect, NS: Not significant.
Table 3. The BHH matrix used to evaluate the effect of 6 factors (A, B, C, D, E and F) on free TPC of PC with R. oryzae in SSF.
Table 3. The BHH matrix used to evaluate the effect of 6 factors (A, B, C, D, E and F) on free TPC of PC with R. oryzae in SSF.
TreatmentABCDEFTPC (mg/g dm)
1−1−1−11114.99 ± 0.24 b
21−1−1−1−113.89 ± 0.25 c
3−11−1−11−12.58 ± 0.11 e
411−11−1−13.37 ± 0.26 d
5−1−111−1−14.15 ± 0.14 c
61−11−11−15.55 ± 0.04 a
7−111−1−114.37 ± 0.38 c
81111114.06 ± 0.57 c
CodeFactor(−1)(1)
ATemperature (°C)3040
BpH56
CInoculum (spo/g)1 × 1061 × 107
DKH2PO4 (g/L)12
EMgSO4·7H2O (g/L)12
F(NH4)2SO4 (g/L)48
Values represent means (n = 3) and mean differences were compared using LSD (p ≤ 0.05). Same letters represent non-significant differences between treatments.
Table 4. Impact of SSF on the physicochemical composition (g/100 g), free TPC and AC of PM.
Table 4. Impact of SSF on the physicochemical composition (g/100 g), free TPC and AC of PM.
ParameterControlFermented
Protein9.03 ± 0.11 b9.94 ± 0.24 a
Total fat2.34 ± 0.16 c4.35 ± 0.16 a
Ash1.32 ± 0.09 b1.69 ± 0.06 a
Crude fiber2.55 ± 0.28 a2.35 ± 0.05 b
Total carb.84.38 ± 0.16 a82.03 ± 0.32 b
Lignin0.15 ± 0.01 a0.15 ± 0.03 a
TPC (mg/g dm)2.40 ± 0.30 b5.55 ± 0.04 a
ABTS (mg TE/g dm)1.08 ± 0.10 b1.70 ± 0.04 a
DPPH (mg TE/g dm)0.61 ± 0.05 b0.93 ± 0.10 a
FRAP (mg Fe2+ /g dm)1.58 ± 0.13 b2.40 ± 0.02 a
Values are means (n = 3) and differences between means were compared using LSD (p ≤ 0.05). Same letters represent non-significant differences between treatments.
Table 5. Identification of compounds in bioprocessed corn with R. oryzae through HPLC-MS.
Table 5. Identification of compounds in bioprocessed corn with R. oryzae through HPLC-MS.
RT m (Min)[M–H]¯Phenolic CompoundsFamilyUFF
m/z
8.97340.9Caffeic acid 4-O-glucosideHydroxycinnamic acidsxx
14.64344.9RosmanolPhenolic terpenesxx
40.12488.9Quercetin 3-O-acetyl-rhamnosideFlavonolsxx
42.75472.9p-Coumaroyl tartaric acid glucosidic esterHydroxycinnamic acidsxx
44.32436.1PhloridzinDihydrochalconesx
47.78185.1PsoralenFuranocoumarins x
49.87713.3Not identifiedxx
51.776172-S-Glutathionyl caftaric acidHydroxycinnamic acidsxx
52.39633.1Galloyl-HHDP-hexosideEllagitanninsx
56.49439.1Not identified xx
58.75161UmbelliferoneHydroxycoumarinsxx
59.11377Oleuropein-aglyconeTyrosolsx
59.86325.1Feruloyl tartaric acidMethoxycinnamic acids x
UF: Unfermented, F: Fermented, x: presence of compounds.
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Ramírez-Esparza, U.; Ordoñez-Cano, A.J.; López-Martínez, L.X.; Espinoza-Hicks, J.C.; Alvarado-González, M.; Ascacio-Valdés, J.A.; Buenrostro-Figueroa, J.J. Solid-State Fermentation with Rhizopus oryzae: Enhancing Antioxidant and Phenolic Content in Pigmented Corn. Resources 2025, 14, 158. https://doi.org/10.3390/resources14100158

AMA Style

Ramírez-Esparza U, Ordoñez-Cano AJ, López-Martínez LX, Espinoza-Hicks JC, Alvarado-González M, Ascacio-Valdés JA, Buenrostro-Figueroa JJ. Solid-State Fermentation with Rhizopus oryzae: Enhancing Antioxidant and Phenolic Content in Pigmented Corn. Resources. 2025; 14(10):158. https://doi.org/10.3390/resources14100158

Chicago/Turabian Style

Ramírez-Esparza, Ulises, Andrés J. Ordoñez-Cano, Leticia X. López-Martínez, José C. Espinoza-Hicks, Mónica Alvarado-González, Juan A. Ascacio-Valdés, and José Juan Buenrostro-Figueroa. 2025. "Solid-State Fermentation with Rhizopus oryzae: Enhancing Antioxidant and Phenolic Content in Pigmented Corn" Resources 14, no. 10: 158. https://doi.org/10.3390/resources14100158

APA Style

Ramírez-Esparza, U., Ordoñez-Cano, A. J., López-Martínez, L. X., Espinoza-Hicks, J. C., Alvarado-González, M., Ascacio-Valdés, J. A., & Buenrostro-Figueroa, J. J. (2025). Solid-State Fermentation with Rhizopus oryzae: Enhancing Antioxidant and Phenolic Content in Pigmented Corn. Resources, 14(10), 158. https://doi.org/10.3390/resources14100158

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