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Article

The Effect of Fermentation with Saccharomyces cerevisiae on the Release of Bound Phenolic Compounds from Wheat Bran and Its Effect on Antioxidant Capacity

by
Víctor Manuel Ocaño-Higuera
1,
Guadalupe López-Avilés
2,
Francisco Javier Almendariz-Tapia
2,
Carmen Lizette Del-Toro-Sánchez
3,
José Agustín Tapia-Hernández
3,
Alba Mery Garzón-García
4,
Octavio Dublán-García
5,
Ariel Alain Vergel-Alfonso
3,
Gustavo Adolfo González-Aguilar
6,
Santiago Valdez-Hurtado
7,
Hebert Jair Barrales-Cureño
8,
José Rogelio Ramos-Enríquez
1,* and
Dalila Fernanda Canizales-Rodríguez
1,*
1
Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Luis Encinas y Rosales, s/n, Hermosillo 83000, Mexico
2
Departamento de Ingeniería Química y Metalurgia, Universidad de Sonora, Luis Encinas y Rosales, s/n, Hermosillo 83000, Mexico
3
Departamento de Investigación y Posgrado en Alimentos (DIPA), Universidad de Sonora, Luis Encinas y Rosales, s/n, Hermosillo 83000, Mexico
4
Ingeniería Industrial, Universidad del Valle, Sede Caicedonia, Caicedonia 762540, Colombia
5
Laboratorio de Alimentos, Facultad de Química, Unidad Cerrillo, Universidad Autónoma del Estado de México, Carretera Toluca-Ixtlahuaca Kilómetro 15.5, El Cerrillo Piedras Blancas, Toluca 50200, Mexico
6
Coordinación de Tecnología de Alimentos de Origen Vegetal, Centro de Investigación en Alimentación y Desarrollo (CIAD), Carretera Gustavo Enrique Astiazarán Rosas, No. 46, Col. La Victoria, Hermosillo 83304, Mexico
7
Unidad Académica Navojoa, Universidad Estatal de Sonora, Blvd, Manlio Fabio Beltrones 810, Col. Bugambilias, Navojoa 85875, Mexico
8
Carrera en Ingeniería en Innovación Agrícola Sustentable, Instituto Tecnológico de Estudios Superiores de Zamora, Tecnológico Nacional de México, Km No. 7, Carretera Zamora-La Piedad, Comunidad El Sáuz de Abajo, Zamora 59720, Mexico
*
Authors to whom correspondence should be addressed.
Processes 2025, 13(11), 3506; https://doi.org/10.3390/pr13113506
Submission received: 30 September 2025 / Revised: 23 October 2025 / Accepted: 27 October 2025 / Published: 31 October 2025
(This article belongs to the Special Issue Green Chemistry: From Wastes to Value-Added Products (2nd Edition))
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Abstract

Wheat bran (WB) is a rich source of phenolic compounds (PCs) with antioxidant capacity (AOX). Approximately 90% of these PCs are bound to the cell wall matrix, which limits their bioavailability. Fermentation with Saccharomyces cerevisiae is an effective strategy to release these bound phenolics. This study aimed to evaluate the effect of fermentation on the release of bound PCs in WB and their AOX during an in vitro digestion system. WB was fermented with Saccharomyces cerevisiae for 2, 4, and 6 days and subsequently subjected to simulated digestion. Free PCs were extracted with methanol, while bound PCs were obtained through alkaline hydrolysis. Total PCs were quantified using the Folin–Ciocalteu method, and AOX was assessed through DPPH, TEAC, and FRAP assays. The content of bound PCs significantly increased after fermentation (p < 0.05): 30.24 ± 0.06 mg GAE/g (day 2), 27.18 ± 0.40 mg GAE/g (day 4), and 28.41 ± 0.40 mg GAE/g (day 6), compared with unfermented WB (7.7 ± 0.21 mg GAE/g) (p < 0.05). AOX was notably enhanced; DPPH reached its peak on day 4 (47.38 ± 0.07 µmol TE/g) (p < 0.05). TEAC was highest on day 2 (26.20 ± 0.43 µmol TE/g) compared with the control (20.14 ± 0.22 µmol TE/g) (p < 0.05) and FRAP showed a slight improvement on day 6 (57.38 ± 0.10 µmol TE/g) relative to the control (56.22 ± 0.13 µmol TE/g) (p < 0.05). Fermentation with Saccharomyces cerevisiae promotes the release of bound PCs in WB and enhances its AOX, highlighting its potential as a functional food ingredient.

1. Introduction

Cereals are among the most important sources of energy, protein, vitamins, and minerals for the global population [1,2]. Within this group, wheat (Triticum aestivum L.) stands out as the most cultivated and consumed grain worldwide [3]. Its nutritional value lies not only in carbohydrates, proteins, lipids, and vitamins, but also in bioactive compounds that confer health-promoting properties. Among these, phenolic compounds (PCs) are particularly relevant due to their diverse biological activities [4]. Beyond its nutritional role, wheat bran represents a major agro-industrial byproduct generated during flour milling. Globally, millions of tons are produced annually, yet a large fraction is used as low value animal feed or discarded. In recent years, the valorization of wheat bran as a source of bioactive compounds has gained attention due to its potential application in functional foods, nutraceuticals, and sustainable ingredients aligned with circular bioeconomy principles [5]. In wheat, most PCs are concentrated in the bran fraction, where approximately 75% occur bound to cell wall constituents such as cellulose, hemicellulose or arabinoxylans, lignin, and proteins [6,7,8,9]. As a result, these bound phenolics are poorly bioaccessible and bioavailable. Hydroxycinnamic acids, especially ferulic acid, are the predominant phenolics in wheat bran, with nearly 90% ester-linked to arabinoxylans and only about 10% present in free or soluble forms [10].
Phenolic compounds are associated with antioxidant, antimicrobial, anticancer, and other bioactivities, with their antioxidant role being the most recognized [11,12,13,14]. By neutralizing free radicals and reactive oxygen species, polyphenols reduce oxidative stress, which is implicated in the development of cardiovascular diseases and other chronic conditions [15,16]. Given their potential health benefits, strategies to release and improve the bioaccessibility of bound phenolics in wheat bran have been widely studied. Various approaches such as alkaline and acid hydrolysis, enzymatic hydrolysis, germination, extrusion, fermentation, and baking have been reported [7,8,9]. Among these, fermentation has emerged as a low-cost, sustainable, and controllable process capable of enhancing phenolic content while preserving antioxidant functionality [17,18]. Saccharomyces cerevisiae, a Generally Recognized as Safe (GRAS) yeast, is widely used in food fermentation and known for producing hydrolytic enzymes (xylanases, esterases, and feruloyl esterases) that facilitate the release of bound phenolics from the cell wall matrix [19].
Previous studies have demonstrated that fermentation enhances the phenolic content and antioxidant activity of cereal matrices; however, most available reports focus on specific microbial strains, short fermentation periods, or omit the integration of in vitro gastrointestinal digestion models to evaluate phenolic bioaccessibility [20,21,22]. Despite these advances, limited information is available on how controlled solid-state fermentation with Saccharomyces cerevisiae affects both the release of free and bound phenolics and their subsequent behavior during digestion. This gap still constrains the full valorization of wheat bran as a functional food ingredient.
Moreover, there is still limited information regarding how controlled solid-state fermentation with Saccharomyces cerevisiae impacts both the release of free and bound phenolics and their subsequent behavior during digestion. In this study, two days of fermentation were identified as the optimal treatment time, providing the highest phenolic content and antioxidant capacity. Although product formulation was beyond the present scope, the fermented wheat bran can be stabilized by drying and stored under vacuum-sealed conditions for later incorporation into functional food prototypes. This finding contributes to bridging the gap between the biochemical understanding of fermentation and its potential technological applications.
Previous research has demonstrated that fermentation can modify the food matrix and enhance the release of phenolic compounds during simulated gastrointestinal digestion. For example, in vitro models have shown that fermentation increases the bioaccessibility and antioxidant activity of cereal-derived phenolics by promoting enzymatic hydrolysis and matrix disintegration [23,24]. These findings support the rationale for combining Saccharomyces cerevisiae fermentation with digestive simulation to assess the functional potential of wheat bran.
Antioxidant activity is one of the main indicators used to evaluate the functional potential of phenolic compounds released during food processing [25]. Their antioxidant potential is commonly assessed through complementary assays based on different reaction mechanisms. The ABTS assay measures the ability of antioxidants to neutralize radical cations via electron and hydrogen atom transfer, while DPPH mainly reflects hydrogen-donating capacity and FRAP evaluates electron-donating power through the reduction of ferric (Fe3+) to ferrous (Fe2+) ions [26]. Each radical shows distinct affinities toward antioxidant molecules, which explains the variability observed among assays. ABTS reacts with both hydrophilic and lipophilic antioxidants through a single electron transfer (SET) mechanism, whereas DPPH preferentially interacts with compounds containing hydroxyl groups near conjugated double bonds, favoring hydrogen atom transfer (HAT) [27]. Together, these methods provide a comprehensive view of redox behavior in complex food matrices [28]. Therefore, evaluating ABTS, DPPH, and FRAP activities in fermented wheat bran helps to identify the specific antioxidant mechanisms enhanced by Saccharomyces cerevisiae fermentation and clarifies how bioprocessing can improve phenolic release, bioaccessibility, and the overall functional value of wheat bran.
In this context, the present study aimed to evaluate the effect of Saccharomyces cerevisiae fermentation on the release of bound phenolic compounds in wheat bran and to determine its impact on antioxidant capacity during an in vitro gastrointestinal digestion system. The novelty of this research lies in demonstrating how controlled fermentation not only enhances the concentration of free and bound phenolics but also improves their bioaccessibility throughout digestion, thereby offering new insights into the design of antioxidant-enriched functional ingredients from wheat bran.

2. Materials and Methods

2.1. Preparation of the Raw Wheat Bran

Wheat bran (variety C.V Kronstad), donated by Molino “La Fama” in Hermosillo, Sonora, Mexico, was used as raw material. The wheat sample was subjected to cleaning following the Reyes-Pérez [29] methodology. Wheat milling was carried out in a Brabender Quadrumant Senior (Brabender Instruments, Duisburg, Alemania) obtaining wheat flour and bran. The wheat bran was subjected to a second grinding in a Laboratory Mill Model 1100 (Thomas Scientific, Swedesboro, NJ, USA), obtaining a particle size of less than 0.05 mm.

2.2. Microbial Fermentation of Wheat Bran

Fermentation was conducted following the methodology of Călinoiu et al. [30] with minor modifications, using Saccharomyces cerevisiae. The yeast employed in this study was a commercial baker’s yeast (Tradi-Pan®, Mexico City, Mexico), corresponding to a domesticated industrial strain of Saccharomyces cerevisiae commonly used in food fermentation. The solid-state fermentation (SSF) was carried out in 500 mL Erlenmeyer flasks containing 25 g of sterile, ground wheat bran (particle size < 1 mm) with a fixed moisture content of 70% (w/w). The substrate was aseptically inoculated with 4.5 mL of yeast suspension (1 × 108 CFU/mL) and 50 mL of sterile distilled water, thoroughly mixed, and incubated under static conditions at 30 °C for up to 6 days. Non-inoculated flasks (54.5 mL of sterile distilled water only) served as controls.
Although pH and microbial viability were not monitored directly, several measures were implemented to ensure that the observed effects were due to active fermentation rather than spontaneous chemical changes. Controlled inoculation and standardized SSF conditions (70% moisture, 30 °C, 6 days) promoted the typical metabolic behavior of Saccharomyces cerevisiae, including CO2 release, ethanol odor development, and time-dependent increases in phenolic and antioxidant contents. These changes are consistent with previously reported yeast-driven SSF in cereal substrates [23,24,30].
For analytical determinations, aliquots of approximately 17 g of culture were withdrawn from the Erlenmeyer flasks every 48 h. The aliquots were inactivated in a convection oven at 60 °C for 24 h and then stored in a desiccator under dark at room-temperature conditions. Dried samples were ground using an electric coffee grinder (Hamilton Beach, 80350R, Glen Allen, VA, USA). Approximately 3.0 g of each ground sample was defatted three times with hexane (1:5 w/v, 5 min per extraction) and air-dried for 48 h under an extraction hood at room temperature.
For the activation, 0.1 g of yeast was weighed and inoculated into 10 mL of potato dextrose broth, previously sterilized (15 min at 121 °C and 15 lb pressure), and then was incubated for 12 h at 30 °C. A cell suspension of 1 × 108 CFU/mL 0.5 McFarland standard solution was prepared. The solid-state fermentation culture was carried out in 500 mL Erlenmeyer flasks containing 25.0 g of the sterile grinded wheat bran, with a fixed humidity of 70% (w/w). The wheat bran samples were aseptically inoculated with 4.5 mL of yeast suspension (1 × 108 CFU/mL) and 50 mL of sterile distilled water, were mixed, and then incubated up to 6 days at 30 °C under static conditions. Samples of the same sterile grinded wheat bran that were treated with this procedure but inoculated with 54.5 mL of only sterile distilled water were used as the control. For the different analyses, aliquots of approximately 17.0 g of culture were withdrawn from the Erlenmeyer flasks at 48 h intervals. The aliquots were inactivated by heat in a convection oven at 60 °C for 24 h, and later were placed in a desiccator under dark conditions at room temperature. The dried samples were ground using an electric coffee grinder (Hamilton Beach, 80350R, Glen Allen, VA, USA). Approximately 3.0 g of each ground sample were transferred to test tubes and defatted three times with hexane (1:5 w/v) for 5 min per extraction. The samples were then air-dried for 48 h under an extraction hood at room temperature.
The extraction of phenolic compounds was carried out under standardized conditions following procedures previously validated for cereal-based matrices. Although no internal standard was used, the method has demonstrated reproducible recoveries (90–95%) in similar wheat bran systems. All extractions were performed in triplicate using identical solvent ratios, agitation speeds, and extraction times to ensure consistency and analytical reliability.

2.3. Free Phenolic Compound Extraction

Free phenolic compounds were extracted according to the method described by Chiremba et al. [31]. For this purpose, 1.0 g of sample (control or fermented wheat bran) was mixed with 15 mL of 80% methanol, sonicated (Branson 1800 Digital Bath, Emerson, St. Louis, MO, USA) for 1 h, and centrifuged (OHAUS-FC5718R 120V, Frontier™ Series 5000 Multi Pro, Parsippany, NJ, USA) for 15 min at 100 rpm. The supernatant was collected in a glass container, and the extraction was repeated three times. The combined supernatants were concentrated under vacuum in a rotary evaporator at 50 °C (DLAB RE 100-Pro, Beijing, China), and the phenolic compounds were re-dissolved in 5 mL of HPLC-grade methanol. The resulting extract was stored in amber vials at −20 °C until analysis. The precipitate obtained during this procedure was reserved for the subsequent extraction of bound phenolic compounds.

2.4. Bound Phenolic Compound Extraction

Bound phenolic compounds were extracted following the method described by Guo and Beta [32]. The precipitate obtained from the previous extraction was dried at 45 °C for 24 h. Subsequently, 100 mg of the dried sample (control and fermented) were mixed with 5 mL of 2 M NaOH for alkaline hydrolysis and sonicated (Branson 1800 Digital Bath, Emerson, St. Louis, MO, USA) for 3 h. The mixture was then acidified with 6 M HCl to a pH of approximately 1.5–2.0. A liquid–liquid extraction was performed with ethyl acetate at twice the volume of the acidified preparation, followed by centrifugation (OHAUS-FC5718R 120V, Frontier™ Series 5000 Multi Pro, Parsippany, NJ, USA) for 15 min at 100 rpm. The supernatant was collected and concentrated under vacuum using a rotary evaporator at 40 °C (DLAB RE 100-Pro, Beijing, China). The residue was re-dissolved in 5 mL of HPLC-grade methanol, and the resulting extract was stored in amber vials at −20 °C until analysis.

2.5. Determination of Total Phenolic Compounds

Total phenolic content was determined using the Folin–Ciocalteu spectrophotometric method described by Singleton and Rossi [33]. This method is based on the reaction of phenolic groups with oxidizing agents. The Folin–Ciocalteu reagent, which contains sodium molybdate and sodium tungstate, reacts with phenolic compounds to form a phosphomolybdic–phosphotungstic complex. In an alkaline medium, this complex undergoes electronic reduction to tungsten oxide (W8O3) and molybdenum oxide (Mo8O23), producing an intense blue color proportional to the number of hydroxyl groups in the phenolic compounds. For analysis, 10 μL of each extract (free and bound phenolics from control and fermented samples) were mixed with 25 μL of 1 M Folin–Ciocalteu reagent and incubated for 5 min at room temperature. Subsequently, 25 μL of 20% Na2CO3 and 140 μL of distilled water were added, and the absorbance was measured at 760 nm using a microplate reader (Thermo Fisher Scientific Inc. Multiskan GO, New York, NY, USA). Results were expressed as mg gallic acid equivalents per gram of sample (mg GAE/g) based on a calibration curve prepared with gallic acid standard solutions ranging from 0.01 to 1.0 mg/mL. This phenolic acid is commonly used as the standard polyphenol because it is a representative natural phenolic found in many plants [34]. All measurements were performed in triplicate.

2.6. Trolox Equivalent Antioxidant Capacity (TEAC) Quantification of Phenolic Compounds

The antioxidant capacity was measured using three methods based on the comparation with a Trolox standard. Trolox, a synthetic water soluble analogue of α-tocopherol, known as the most active form of vitamin E, serves as the reference compound for expressing results as Trolox equivalent antioxidant capacity (TEAC), which allows the reliable comparison of antiradical activity among samples [35]. These methods are the ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) decolorization, the DPPH (1,1-difenil-2-picrihidrazil), and the ferric-reducing ability of plasma (FRAP) assays [36,37,38].

2.6.1. ABTS Decolorization Assay

Antioxidant activity was determined using the ABTS+ radical cation decolorization assay described by Re et al. [36]. This method is based on the quantification of ABTS+ discoloration resulting from its reduction to ABTS by antioxidant compounds. The ABTS+ radical, which absorbs at 734 nm, exhibits a bluish green coloration. To prepare the radical solution, 0.0378 g of K2S2O8 were dissolved in 1 mL of distilled water, and 88 μL of this solution were added to an ABTS solution prepared by dissolving 19.3 mg of ABTS in 5 mL of distilled water. The mixture was kept in the dark at room temperature for 14 h to generate the ABTS+ radical. After incubation, 1 mL of the radical solution was diluted with 88 mL of ethanol to obtain an absorbance of 0.70 ± 0.01 at 734 nm. For the assay, 270 μL of the ABTS+ solution were mixed with 20 μL of sample (control and fermented wheat bran) and incubated for 30 min in the dark. Absorbance was then measured at 734 nm. Results were expressed as micromoles of Trolox equivalents per gram of sample (μmol TE/g) based on a calibration curve constructed with Trolox dissolved in methanol. All measurements were performed in triplicate.

2.6.2. DPPH Assay

This method is based on the reaction of the radical DPPH (1,1-difenil-2-picrihidrazil), which presents a violet coloration in alcoholic solution, with other radical or a hydrogen atom, producing the reduced form DPPH-R or DPPH-H with the simultaneous loss of the violet coloration; the measurement of this coloration is performed at 515 nm. In the present research, the method described by Molyneux [37] was followed. For this, 2.5 mg of radical DPPH were dissolved in 50 mL of methanol, which gives an absorbance of 0.7 ± 0.01 at 515 nm. A volume of 220 μL of this solution was mixed with 20 μL of sample (control and fermented wheat bran) and it was left to react for 30 min in the dark; afterwards, the absorbance was measured at 515 nm. Results were expressed as micromoles of Trolox equivalents per gram (μmolTE/g) of sample from a calibration curve using Trolox dissolved in methanol. Each sample was studied by triplicate.

2.6.3. FRAP Assay

Ferric ion reducing antioxidant power (FRAP), also known as the FRAP assay, was determined following the methodology described by Rubio et al. [38] and referenced by Rioja-Antezana et al. [39]. This method is based on the reduction of ferric ion (Fe3+) present in the FRAP reagent to ferrous ion (Fe2+) by the action of antioxidants. The FRAP reagent is composed of acetate buffer (300 mmol/L, pH 3.6), TPTZ (2,4,6-tris(2-pyridyl)-s-triazine, 40 mmol/L prepared in 40 mmol/L HCl), and FeCl3 (20 mmol/L). When the reagent comes into contact with a sample, the ferrous ion forms a ferrous–TPTZ complex (TPTZ–Fe2+) with a blue coloration proportional to the reducing capacity of the sample. Quantification is performed by measuring the absorbance at 593 nm and comparing the values with a FeSO4 standard curve.
In the present research, two stock solutions were first prepared: (1) an acetate buffer solution (300 mmol/L, pH 3.6) and (2) a solution containing TPTZ (40 mmol/L) and FeCl3 (20 mmol/L) in 40 mmol/L HCl. The FRAP reagent was then freshly prepared by mixing CH3COONa (pH 3.6), TPTZ, and FeCl3. A volume of 280 μL of this reagent was mixed with 20 μL of sample (control and fermented wheat bran) and allowed to react for 30 min in the dark. Afterwards, the absorbance was measured at 638 nm using a microplate reader. Results were expressed as micromoles of Trolox equivalents per gram of sample (μmol TE/g) based on a calibration curve prepared with Trolox dissolved in methanol at concentrations ranging from 0.01 to 1.0 mg/mL. Each sample was analyzed in triplicate.

2.7. Evaluation of the Release of Phenolic Compounds with an In Vitro Gastrointestinal System

An in vitro gastrointestinal system simulates gastrointestinal digestion through specific enzymes of the different digestion phases. The α-amylase enzyme or human saliva at pH of 7.0 is used in the oral phase. Human saliva was collected exclusively for analytical purposes from healthy adult volunteers belonging to the research group. The procedure did not involve the collection of personal data, medical intervention, or any potential risk to participants and therefore did not require formal ethics committee approval, in accordance with institutional and national guidelines for minimal-risk research. All donors voluntarily provided saliva samples after being informed of the experimental purpose, following the ethical principles of the Declaration of Helsinki and the MDPI Policy on Research Involving Humans. The same group of volunteers was used for all assays to ensure consistency and reproducibility.
The gastric phase is simulated using pepsin enzyme at pH of 2.0. And the pancreatin enzyme at pH of 7.0 is used the intestinal phase. In the present research, the methodology described by Pérez-Pérez et al. [40] was followed, with slight modifications. First, 1.0 g of sample (control and fermented wheat bran) was chewed by health volunteers for 15 min, for 15 times (before, they washed their teeth and did not ingested food at least 90 min previous the experiment). The chewed sample was homogenized with 10 mL of distilled water and acidified with HCl 6 M to achieve a pH of 1.5-2.0. Later, 22.5 mL of pepsin (315 U/mL prepared in 0.01 M phosphate buffered saline (PBS) buffer) and 22.5 mL of distilled water were added to the acidified chewed sample, and it was incubated (Wise Bath, DAIHAN Scientific, WSB-18, Wonju, South Korea) for 2 h at 37 °C under 80 rpm stirring. Afterwards, the sample was neutralized with NaOH 1.0 M, and 5.625 mL of pancreatin (4 mg/mL prepared in 0.01 M PBS) were added and mixed. The sample was later added to a 14,000 Da dialysis membrane (Sigma-Aldrich D9888-100FT, St. Louis, MO, USA). The membrane was placed inside a glass vessel with 160 mL of 1 M PBS and was incubated for 2 h at 37 °C under 80 rpm stirring. The antioxidant capacity in each in vitro gastrointestinal phase was measured with ABTS, DPPH, and FRAP assays previously described.

2.8. Experimental Design and Statistical Analysis

The experimental design consisted of a solid-state fermentation of wheat bran using Saccharomyces cerevisiae at 30 °C for 0 (control), 2, 4, and 6 days, with three independent replicates per treatment. Fermented and unfermented samples were subjected to sequential extraction to obtain free and bound phenolic compounds, followed by determination of total phenolics and antioxidant capacity through Folin–Ciocalteu, DPPH, ABTS, and FRAP assays, as well as evaluation under an in vitro gastrointestinal digestion model. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis of variance (ANOVA), and mean comparisons were carried out with Tukey’s test to identify significant differences among treatments. A significance level of p < 0.05 was applied to all tests to determine the effects of fermentation time on phenolic content and antioxidant activity.
All experimental data were obtained from three independent biological replicates, each corresponding to a separate fermentation performed under identical conditions. Each analytical determination (phenolic content and antioxidant assays) was performed in technical triplicates, and mean values were used to calculate the corresponding biological replicate averages. Standard deviation (SD) values therefore represent biological variability among independent fermentations.

3. Results and Discussion

3.1. Free and Bound Phenolic Compounds Determination

Wheat bran constitutes the outer layers of the grain obtained during the milling process, accounting for 14–19% of the total grain weight. Its chemical composition depends on the milling degree [41]. Typically, wheat bran contains 43–60% non-starch polysaccharides, 11–24% starch, 14–20% proteins, 3–4% lipids, and 3–8% minerals [42]. The non-starch polysaccharide fraction is mainly composed of arabinoxylans (70%), cellulose (19%), lignin, and β-glucans (6%) [43], forming a complex structural matrix that retains bioactive compounds such as phenolic acids, flavonoids, tocopherols, and phytosterols. The majority of phenolic acids, mainly ferulic acid, present in wheat bran are esterified to cell wall polysaccharides, which limits its release and bioavailability [44]. Understanding this composition and structure is essential to explain the biochemical effects of fermentation and its impact on the release of phenolic compounds and the antioxidant capacity of wheat bran [45].
The concentrations of free and bound phenolic compounds in the control wheat bran were 1.400 ± 0.042 and 7.793 ± 0.214 mg GAE/g, respectively, resulting a bound-to-free phenolic ratio of approximately 5.5:1.0. After fermentation, both fractions increased markedly (Table 1). Bound phenolics rose by nearly fourfold, whereas free phenolics increased up to sixfold. Remarkably, two days of fermentation were sufficient to reach the maximum concentration of both free and bound phenolics. At this point, total phenolic content increased by 422% relative to the unfermented control, exceeding the 112% increase reported by Călinoiu [30] after three days of Saccharomyces cerevisiae fermentation. After four days, a slight additional rise in free phenolics was detected, whereas bound phenolics showed a moderate decline. The statistical lettering confirmed significant variations (p < 0.05) in total phenolic content among treatments, with the two-day fermentation exhibiting the highest overall value, consistent with the enzymatic release of bound phenolics during the early fermentation stage.
The sharp increase in phenolic content can be attributed to enzymatic activities generated during fermentation, likely associated with enzymes such as xylanases, esterases, and feruloyl esterases, which cleave ester or ether linkages between phenolic acids and arabinoxylan backbones [46]. Solid-state fermentation provides favorable conditions for the production of these enzymes, facilitating the breakdown of hemicellulose and lignin structures and promoting phenolic release [18,47,48]. Recent studies confirm similar patterns: Saccharomyces cerevisiae fermentation of wheat bran significantly increased extractable phenolic acids and antioxidant activity within 48 h, while co-fermentation with lactic acid bacteria further boosted free phenolic content and antioxidant potential [6,49].
However, specific enzyme activities were not determined in this study, and the interpretation is based on well-documented evidence of hydrolytic enzyme production by yeast and related microorganisms in cereal-based fermentations. Future research should aim to identify and quantify these enzymatic systems to confirm their contribution to phenolic release.
Compared with other processing technologies, our fermentation approach proved particularly effective. For example, Ramos-Enríquez et al. [50] observed only a 43% increase in total phenolics after extrusion of wheat bran, far below the >400% increase achieved here after two days of fermentation. These findings highlight the advantage of controlled microbial fermentation over purely thermal or mechanical treatments in enhancing phenolic bioacessibility. The separate evaluation of free and bound phenolic fractions was carried out to better understand the redistribution of phenolics during fermentation. Free phenolics correspond to soluble compounds easily released from the matrix, whereas bound phenolics remain esterified or ether-linked to cell wall polymers. Their joint analysis provides complementary insight into the enzymatic hydrolysis processes and the total antioxidant potential of fermented wheat bran.

3.2. ABTS Decolorization Assay

Table 2 shows the ABTS radical scavenging activity of free, bound, and total phenolic fractions from control and fermented wheat bran. In all cases, the antioxidant capacity was roughly ten times higher in the bound fraction compared with the free fraction, highlighting the dominant contribution of bound phenolics to the overall antioxidant potential of wheat bran. Fermentation for 2 and 4 days yielded the highest total ABTS values, with increases of 30% and 28%, respectively, relative to the unfermented control, whereas a six-day fermentation resulted in only a modest 9% gain.
This trend mirrors the dynamic release of phenolic compounds during fermentation, which is observed in Figure 1. During the first 48–72 h, Saccharomyces cerevisiae produces hydrolytic enzymes (xylanases, feruloyl esterases, β-glucosidases) that cleave phenolics from the arabinoxylan matrix, thereby enhancing radical-scavenging capacity [45,51]. After 3 to 4 days, nutrient depletion and accumulation of inhibitory metabolites likely slow enzymatic activity and phenolic release, explaining the decline observed at 6 days [30,52]. Similar time-dependent patterns have been reported in other cereal fermentations, where ABTS activity peaks early and then plateaus or decreases with extended incubation [49,53].
The ABTS values obtained after 2 and 4 days are comparable to those reported for fermented red wheat [54] and exceed those achieved by extrusion alone, which typically yields more modest improvements in antioxidant capacity [50]. These findings reinforce the advantage of microbial fermentation over purely thermal processing for enhancing the radical-scavenging potential of wheat bran.

3.3. DPPH Assay

Table 3 summarizes the antioxidant capacity measured by the DPPH radical scavenging assay for free, bound, and total phenolic fractions of control and fermented wheat bran. Similar to the ABTS results, the antioxidant capacity of the bound phenolic fraction was nearly ten times higher than that of the free fraction in all fermented samples. In contrast, the control wheat bran showed a lower bound-to-free ratio (≈5.6:1), highlighting the strong impact of fermentation on the liberation of phenolic compounds with high radical scavenging activity.
The highest total DPPH values were recorded after 2 and 4 days of fermentation, with increases of 20% and 25%, respectively, compared to the control. After 6 days, the total antioxidant capacity increased by only ~2%, reflecting a decline in enzyme activity and possible depletion of fermentable substrates. Interestingly, while the ABTS assay peaked slightly at day 2, the DPPH assay showed its maximum at day 4 (Figure 1), suggesting that different antioxidant mechanisms (electron transfer vs. hydrogen atom donation) may respond differently to the phenolic profile generated during fermentation [51].
These observations are in line with recent findings that Saccharomyces cerevisiae fermentation enhances the production of low-molecular-weight phenolics capable of effectively reducing the DPPH radical [49]. Also, results obtained with DPPH assay are similar to those reported by Sandu [52] using Aspergillus awamori Nakazawa for the fermentation. Similar time-dependent patterns—rapid increase followed by stabilization or decline—have been reported in wheat and barley fermentations, where the early production of hydrolytic enzymes such as xylanases and feruloyl esterases leads to a burst of antioxidant release that diminishes as nutrients become limiting [45].
Furthermore, the DPPH values obtained in this study surpass those reported for wheat bran fermented with Bifidobacterium spp. [55] and are markedly higher than those achieved by extrusion processing [48]. This reinforces the advantage of controlled yeast fermentation over bacterial fermentation or mechanical treatments for maximizing antioxidant activity.

3.4. FRAP Assay

Table 4 presents the ferric-reducing antioxidant power (FRAP) of free, bound, and total phenolic fractions in control and fermented wheat bran. Unlike the ABTS and DPPH assays, FRAP values showed only minor fluctuations throughout fermentation. Although slight increases and decreases were observed over time, no significant decline was detected at six days. In fact, a modest increase in total FRAP values was recorded in the six-day fermented samples compared with the control, indicating that the reducing capacity of the phenolic compounds remained stable or slightly improved even after prolonged incubation (Figure 1).
This behavior contrasts with the sharper decreases observed in ABTS and DPPH activities at day six, suggesting that the redox potential of certain phenolic constituents particularly those responsible for electron transfer—remains intact despite the possible degradation of more labile compounds. Similar stability of FRAP activity during yeast fermentation of cereals has been reported previously by Đorđević [55] and confirmed in recent studies of Saccharomyces cerevisiae fermentation of wheat bran and barley [45,49]. These findings indicate that while hydrogen-donating capacity (DPPH) and radical-cation scavenging (ABTS) may decline with extended fermentation, the ferric-reducing ability can remain unaffected or even slightly enhanced, likely due to the formation of more stable phenolic derivatives or Maillard reaction products with strong reducing potential [51]. Overall, the FRAP results demonstrate that fermentation by Saccharomyces cerevisiae maintains or slightly enhances the electron-donating capacity of wheat bran phenolics, underscoring the robustness of the antioxidant system generated through solid-state fermentation.

3.5. Evaluation of the Release of Phenolic Compounds with an In Vitro Gastrointestinal System

The total phenolic content and antioxidant activities (FRAP, ABTS and DPPH) obtained during the successive phases of the in vitro gastrointestinal digestion are shown in Figure 2a–c. For the control wheat bran, a progressive increase in total phenolic compounds was observed across the three digestion stages, with the highest concentration in the intestinal phase (pancreatin) and the lowest in the oral phase (saliva). Antioxidant activity followed the same trend in all assays (intestine > stomach > mouth). The first marked rise occurred in the gastric stage, where the acidic conditions and pepsin activity promote hydrolysis of peptide chains and disruption of carbohydrate linkages, thereby enhancing the release of phenolic compounds [23].
During the intestinal stage, pancreatin further hydrolyzed the residual food matrix, producing the greatest increase in total phenolic content (Figure 2d). This pronounced rise indicates that the longer exposure time and the alkaline environment of the intestinal phase were sufficient to break most covalent bonds within the matrix, liberating nearly all bound phenolics. These findings confirm that the digestive process plays a key role in releasing phenolic compounds initially trapped in the wheat bran cell wall.
Fermented wheat bran samples displayed the same upward trend, but with significantly higher phenolic concentrations than the control at every digestion stage. Among the fermented samples, the highest total phenolic release was obtained after two days of fermentation, aligning with the earlier fermentation results. Antioxidant activity also increased progressively across the digestive phases, with fermented samples outperforming the control in all assays except DPPH.
The intestinal stage showed the greatest enhancement in antioxidant capacity. This improvement can be attributed to the transition from acidic to alkaline conditions, which favors deprotonation of phenolic hydroxyl groups and thereby strengthens electron-donating and radical-scavenging activities [24]. The slightly lower DPPH values observed in fermented samples may reflect the pH sensitivity of the DPPH radical, which is less stable under neutral or alkaline conditions [56], particularly in the saliva and gastric phases. Overall, these results demonstrate that Saccharomyces cerevisiae fermentation not only increases the pool of phenolic compounds prior to digestion but also enhances their bioaccessibility during gastrointestinal transit, which is critical for maximizing their potential health benefits.

4. Conclusions

This study demonstrated that fermentation of wheat bran with Saccharomyces cerevisiae effectively promotes the release of bound phenolic compounds and enhances antioxidant capacity. The highest improvement in phenolic content was observed after two days of fermentation, with significant increases in both free and bound phenolics compared to unfermented wheat bran. Antioxidant activity, evaluated by DPPH, TEAC, and FRAP assays, also showed notable improvements, particularly after two and four days of fermentation. Furthermore, in vitro gastrointestinal digestion confirmed that fermentation enhanced the release and bioaccessibility of phenolic compounds, with the intestinal phase showing the greatest impact. These findings highlight fermentation with Saccharomyces cerevisiae as an accessible and sustainable strategy to increase the functional value of wheat bran, supporting its potential use as an ingredient in the development of antioxidant-enriched functional foods. Future studies should address the optimization of fermentation parameters and evaluate the bioavailability of these compounds in vivo to strengthen their application in preventive nutrition.

Author Contributions

Conceptualization, V.M.O.-H. and D.F.C.-R.; methodology, V.M.O.-H., G.L.-A., F.J.A.-T., C.L.D.-T.-S., J.A.T.-H. and H.J.B.-C.; validation, V.M.O.-H., F.J.A.-T., C.L.D.-T.-S., J.A.T.-H., O.D.-G., G.A.G.-A., H.J.B.-C. and J.R.R.-E.; formal analysis, G.L.-A., F.J.A.-T., A.M.G.-G., A.A.V.-A. and S.V.-H.; investigation, G.L.-A., J.A.T.-H., A.M.G.-G., O.D.-G., S.V.-H. and H.J.B.-C.; resources, C.L.D.-T.-S., J.A.T.-H., O.D.-G. and G.A.G.-A.; data curation, G.L.-A., A.M.G.-G., A.A.V.-A. and S.V.-H.; writing—original draft preparation, V.M.O.-H. and D.F.C.-R.; writing—review & editing, F.J.A.-T., C.L.D.-T.-S., A.M.G.-G., A.A.V.-A., G.A.G.-A., H.J.B.-C. and J.R.R.-E.; visualization, O.D.-G., A.A.V.-A. and S.V.-H.; supervision, G.A.G.-A., J.R.R.-E. and D.F.C.-R.; project administration, V.M.O.-H., J.R.R.-E. and D.F.C.-R.; funding acquisition, J.R.R.-E. and D.F.C.-R. 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.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
WB wheat bran
AOX antioxidant capacity
PCs phenolic compounds
ABTS2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)
TEACTrolox equivalent antioxidant capacity
FRAPferric-reducing ability of plasma
SDstandard deviation

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Processes 13 03506 g001
Figure 1. Changes in antioxidant capacity ABTS, DPPH, FRAP and total phenolic content of wheat bran during Saccharomyces cerevisiae fermentation. Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Figure 1. Changes in antioxidant capacity ABTS, DPPH, FRAP and total phenolic content of wheat bran during Saccharomyces cerevisiae fermentation. Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Processes 13 03506 g001
Processes 13 03506 g002
Figure 2. Antioxidant capacity FRAP (a), ABTS (b), DPPH (c), and total phenolic content (d) of wheat bran during the oral, gastric, and intestinal stages of an in vitro gastrointestinal model. Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Figure 2. Antioxidant capacity FRAP (a), ABTS (b), DPPH (c), and total phenolic content (d) of wheat bran during the oral, gastric, and intestinal stages of an in vitro gastrointestinal model. Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Processes 13 03506 g002
Table 1. Phenolic compound concentration as determined by the Folin–Ciocalteu method in the control and fermented wheat bran.
Table 1. Phenolic compound concentration as determined by the Folin–Ciocalteu method in the control and fermented wheat bran.
Fermentation Time (Days)Free Phenolic Compounds (mgGAE/g)Bound Phenolic
Compounds (mgGAE/g)		Total Phenolic
Compounds (mgGAE/g)
0 (Control)1.40 ± 0.04 a7.79 ± 0.2 b9.19 ± 0.13 c
28.56 ± 0.05 d30.24 ± 0.06 e38.81 ± 0.06 f
49.55 ± 0.01 g27.18 ± 0.40 h36.73 ± 0.21 i
65.46 ± 0.00 j28.41 ± 0.40 k33.87 ± 0.20 l
Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Table 2. Antioxidant capacity quantification of phenolic compounds as determined by the ABTS assay in the control and fermented wheat bran.
Table 2. Antioxidant capacity quantification of phenolic compounds as determined by the ABTS assay in the control and fermented wheat bran.
Fermentation Time (Days)Free Phenolic Compounds (μmolTE/g)Bound Phenolic Compounds (μmolTE/g)Total Phenolic
Compounds (μmolTE/g)
0 (Control)2.08 ± 0.00 a20.14 ± 0.22 b22.22 ± 0.11 c
22.61 ± 0.00 d26.20 ± 0.43 e28.81 ± 0.22 f
42.54 ± 0.03 g25.93 ± 0.96 h28.47 ± 0.50 i
62.56 ± 0.04 j21.75 ± 0.46 k24.31 ± 0.25 l
Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Table 3. Antioxidant capacity quantification of phenolic compounds as determined by the DPPH assay in the control and fermented wheat bran.
Table 3. Antioxidant capacity quantification of phenolic compounds as determined by the DPPH assay in the control and fermented wheat bran.
Fermentation Time (Days)Free Phenolic
Compounds (μmolTE/g)		Bound Phenolic
Compounds (μmolTE/g)		Total Phenolic
Compounds (μmolTE/g)
0 (Control)6.36 ± 0.18 a35.54 ± 0.11 b41.91 ± 0.15 c
24.66 ± 0.05 d45.72 ± 0.02 e50.38 ± 0.03 f
45.12 ± 0.06 g47.38 ± 0.07 h52.50 ± 0.06 i
64.57 ± 0.00 j38.11 ± 0.01 k42.69 ± 0.01 l
Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
Table 4. Antioxidant capacity quantification of phenolic compounds as determined by the FRAP assay in the control and fermented wheat bran.
Table 4. Antioxidant capacity quantification of phenolic compounds as determined by the FRAP assay in the control and fermented wheat bran.
Fermentation Time (Days)Free Phenolic
Compounds (μmolTE/g)		Bound Phenolic
Compounds (μmolTE/g)		Total Phenolic
Compounds (μmolTE/g)
0 (Control)10.56 ± 1.65 ai56.22 ± 0.13 b66.78 ± 0.89 c
211.87 ± 0.03 a56.59 ± 0.07 d68.46 ± 0.05 e
413.02 ± 0.04 f48.43 ± 0.07 g61.45 ± 0.06 h
611.69 ± 0.12 i57.38 ± 0.10 j69.06 ± 0.11 k
Data represent the mean ± SD of n = 3. Different lowercase letters indicate statistically significant differences (p < 0.05).
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Ocaño-Higuera, V.M.; López-Avilés, G.; Almendariz-Tapia, F.J.; Del-Toro-Sánchez, C.L.; Tapia-Hernández, J.A.; Garzón-García, A.M.; Dublán-García, O.; Vergel-Alfonso, A.A.; González-Aguilar, G.A.; Valdez-Hurtado, S.; et al. The Effect of Fermentation with Saccharomyces cerevisiae on the Release of Bound Phenolic Compounds from Wheat Bran and Its Effect on Antioxidant Capacity. Processes 2025, 13, 3506. https://doi.org/10.3390/pr13113506

AMA Style

Ocaño-Higuera VM, López-Avilés G, Almendariz-Tapia FJ, Del-Toro-Sánchez CL, Tapia-Hernández JA, Garzón-García AM, Dublán-García O, Vergel-Alfonso AA, González-Aguilar GA, Valdez-Hurtado S, et al. The Effect of Fermentation with Saccharomyces cerevisiae on the Release of Bound Phenolic Compounds from Wheat Bran and Its Effect on Antioxidant Capacity. Processes. 2025; 13(11):3506. https://doi.org/10.3390/pr13113506

Chicago/Turabian Style

Ocaño-Higuera, Víctor Manuel, Guadalupe López-Avilés, Francisco Javier Almendariz-Tapia, Carmen Lizette Del-Toro-Sánchez, José Agustín Tapia-Hernández, Alba Mery Garzón-García, Octavio Dublán-García, Ariel Alain Vergel-Alfonso, Gustavo Adolfo González-Aguilar, Santiago Valdez-Hurtado, and et al. 2025. "The Effect of Fermentation with Saccharomyces cerevisiae on the Release of Bound Phenolic Compounds from Wheat Bran and Its Effect on Antioxidant Capacity" Processes 13, no. 11: 3506. https://doi.org/10.3390/pr13113506

APA Style

Ocaño-Higuera, V. M., López-Avilés, G., Almendariz-Tapia, F. J., Del-Toro-Sánchez, C. L., Tapia-Hernández, J. A., Garzón-García, A. M., Dublán-García, O., Vergel-Alfonso, A. A., González-Aguilar, G. A., Valdez-Hurtado, S., Barrales-Cureño, H. J., Ramos-Enríquez, J. R., & Canizales-Rodríguez, D. F. (2025). The Effect of Fermentation with Saccharomyces cerevisiae on the Release of Bound Phenolic Compounds from Wheat Bran and Its Effect on Antioxidant Capacity. Processes, 13(11), 3506. https://doi.org/10.3390/pr13113506

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