Journal of biological and health sciences http://biotecnia.unison.mx
Universidad de Sonora
ISSN: 1665-1456
Original Article
1 Facultad de Ciencias de la Nutrición y Gastronomía, Universidad Autónoma de Sinaloa, Av. Cedros S/N y Calle Sauces, Los Fresnos Fracc, C.P. 80019 Culiacán Rosales, Sinaloa, México.
2 Programa de Posgrado Integral en Biotecnología, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Ciudad Universitaria, Culiacán, Sinaloa, C.P. 80030, México.
3 Maestría en Ciencia y Tecnología de Alimentos, Facultad de Ciencias Químico Biológicas, Universidad Autónoma de Sinaloa, Ciudad Universitaria, Culiacán, Sinaloa, C.P. 80030, México.
Efecto del Proceso de Nixtamalización Tradicional y Extrusión sobre la Bioaccesibilidad y Actividad Antioxidante de Compuestos Fenólicos de Tortillas de Maíz Azul Durante la Fermentación In Vitro por la Microbiota Colónica Humana
In recent years, tortillas made with pigmented maize have garnered interest due to their contribution of polyphenols, considered natural antioxidant compounds with antihy- pertensive, antidiabetic, and anti-carcinogenic properties. In maize, the greatest contribution of polyphenols is in in- soluble form. These secondary metabolites are released by the colonic microbiota making them more bioaccessible to the organism. In the present work, chemical composition, bioaccessibility, release of phenolic compounds, and anti- oxidant activity by colonic microbiota in tortillas, made with nixtamalized (NMT) and extruded (EMT) blue maize, were evaluated and compared. EMT had higher protein, lipid, and ash content than NMT. However, NMT had higher anthocy- anin content than its EMT counterpart (4.01 and 2.28 mg CGE/100 g, dw). The bound phenolic fraction in both tortillas represents > 80 %. At 5 h of in vitro fermentation by colonic microbiota, NMT showed the highest average in phenolic release, bioaccessibility, and antioxidant activity [11.78 mg GAE/ g, dw, 88.23 %, and 569.82 (ORAC) and 26.76 (ABTS)
µmol TE/g] than EMT. The use of traditional nixtamalization to produce maize tortillas will continue to be the main pro- cess that brings health benefits to consumers.
En los últimos años las tortillas elaboradas con maíces pig- mentados han despertado interés debido al aporte de poli- fenoles, los cuales son considerados antioxidantes naturales y poseen propiedades antihipertensivas, antidiabéticas, y anticarcinogénicas. En maíz, el mayor aporte de polifenoles se encuentra en forma insoluble. Estos metabolitos secun- darios son liberados por la microbiota del colón haciéndolos
*Autor para correspondencia: Sara Monzerrat Ramírez Olvera Correo-e: smora@uas.edu.mx
Received: November 24, 2024
Accepted: January 7, 2025
Published: February 7, 2025
más bioaccesibles para el organismo. En el presente trabajo se evaluó y comparó composición química, bioaccesibilidad, liberación de compuestos fenólicos y actividad antioxidante por la microbiota colónica en tortillas elaboradas con maíz azul nixtamalizado (NMT) y extrudido (EMT). Las EMT pre- sentaron mayor contenido de proteína, lípidos y cenizas, con respecto NMT. Sin embargo, NMT presentó mayor contenido de antocianinas que su contraparte EMT (4.01 y 2.28 mg ECG/100 g, bs). La fracción de fenólicos ligados en ambas tortillas representa > 80 %. A las 5 h de fermentación in vitro por la microbiota del colon, NMT mostró el mayor promedio de liberación y bioaccesibilidad de fenólicos, y actividad antioxidante [11.78 mg EAG/g, bs, 88.23 %, y 569.82 (ORAC) y 26.76 (ABTS) µmol ET/g] que EMT. El proceso de nixtamali- zación tradicional para elaborar tortillas seguirá siendo clave importante para el aporte de beneficios en la salud de los consumidores.
Nowadays, products elaborated with pigmented maize, such as blue maize tortillas, have received considerable attention from a health benefit perspective due to their high content of secondary metabolites, such as phenolic compounds, an- thocyanins, and carotenoids (Mora-Rochin et al., 2016; Colín- Chávez et al., 2020). These compounds have been shown to possess potential for the prevention of chronic noncommu- nicable diseases, such as cancer, arterial hypertension, and diabetes, through their antioxidant capacity (Mora-Rochin et al., 2019; Domínguez-Hernández et al., 2022). However, this property, provided by these secondary metabolites or phytochemicals present in maize kernels, depends not only on their concentration but also on their bioaccessibility after
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DOI: doi.org/10.18633/biotecnia.v27.2520
ingestion (Astorga-Gaxiola et al., 2023; Menchaca-Armenta et al., 2023).
Maize in Mexico is processed mainly by the ancestral method inherited from the Aztecs, nixtamalization, where the maize kernel is put in water with lime, cooked at high temperatures, and left to stand for long periods of time. These characteristics of the process significantly change the nutritional content, concentration of phytochemicals, antioxidant properties, as well as the bioaccessibility of different biomolecules (Enriquez-Castro et al., 2020). On the other hand, grain cooking using extrusion technology has been used as an effective processing method to improve the nutritional quality of cereals and legumes, where it has been reported that extrusion modifies physicochemical properties of insoluble fiber, resulting in an increase in soluble fiber due to the shearing forces. Additionally, protein inhibitors and other anti-nutritional factors (trypsin inhibitors, tannins, oxalates, and lectins) are inactivated or denatured, and it has been reported that extrusion significantly modifies the con- tent of phytochemicals with antioxidant properties (Choton et al., 2020; Kamau et al., 2020; Bonilla-Vega et al., 2022).
In maize, phenolic compounds are bound to cell wall structures, mainly in the pericarp (> 80 %), which inhibits their digestion in the upper part of the gastrointestinal tract (Méndez-Lagunas et al., 2020; Astorga-Gaxiola et al., 2023). Thus, upon reaching the colon, these compounds are me- tabolized by the colonic microbiota, allowing their release, and their conversion into smaller secondary metabolites and subsequent absorption, achieving local and systemic beneficial effects (Tomás-Barberán and Espín, 2019; Astor- ga-Gaxiola et al., 2023). Phenolic compounds regulate the composition and functionality of gut microbiota, which can have significant implications for human health. Gut bacteria perform various essential roles, including food fermentation, pathogen defense, immune system stimulation, and vitamin synthesis. Foods rich in phenolic compounds may exhibit effects comparable to those of classical prebiotics. Addition- ally, the metabolites derived from phenolic transformation contribute to regulating redox balance and metabolism, while demonstrating antioxidant, antimicrobial, anti-inflam- matory, and anticancer properties (Hou et al., 2022; Kwon et al., 2024).
It is important to mention that, to date, there is little scientific evidence in regard to the effect of the different pro- cesses for the elaboration of blue maize tortillas on the struc- ture of the food matrix that facilitates the metabolization of phenolic compounds by the colonic microbiota. Therefore, the objective of the present work is to evaluate and compare the bioaccessibility, the release of phenolic compounds by the colonic microbiota, and antioxidant activity in blue maize tortillas made by the ancestral process of nixtamalization and by the extrusion process.
Whole blue maize was sourced from a local market in Cu- liacán, Sinaloa, Mexico. Seeds were cleaned and stored in airtight containers at 4 °C until use.
The flour was obtained with the procedure described by Mo- ra-Rochín et al. (2010). Batches of 100 g of blue maize were cooked for 30 min in a lime solution [5.4 g Ca (OH)₂/L distilled water] at 85 °C, using a grain to water ratio of 1:3 (w/v) and set to stand for 8 h. After the setting period, the cooking liquid (nejayote) was removed, and the nixtamalized grain was washed under running water to remove excess lime and pericarp. The nixtamal was dried at 55 °C for 12 h in a forced air oven. After cooling for 30 min at room temperature, it was ground using a cyclone mill with an 80 mesh (0.180 mm) sieve. The resulting flour was stored at 4 °C until use.
The methodology proposed by Milán-Carrillo et al. (2006) was followed. Briefly, 500 g of blue maize seeds were ground and passed through a 40 US mesh (0.074 mm) sieve. The resulting flour was mixed with lime (0.21 g Ca (OH)2/100 g flour). H2O was added until 28 % moisture was achieved. The extrusion cooking process was conducted using a 20 DN ex- truder (CW Brabender Instruments, Inc, NJ, USA) with a single strand of 19 mm in diameter. The operating conditions were: a temperature of 85 °C and a screw speed of 240 rpm. The extrudates were cooled to room temperature and ground to 80-US mesh (0.180 mm). Finally, everything was stored in polyethylene bags at 4 °C until further use.
The protocol described by Mora-Rochín et al. (2016) was followed. Four hundred g of flour and 400 mL of water were mixed until a dough of suitable consistency was obtained. Small portions of dough (30 g) were pressed and shaped into flat discs (15 cm) using a manual press (Casa Herrera, Mexico DF, Mexico). The disks were baked on a hot griddle (270 ± 10
°C) for 15 s on one side and 30 s on the other side. Finally, the first side was cooked again until a puffed tortilla was ob- served. The fresh tortillas were dried and ground (UD Cyclone Sample Mill, UD Corp. Boulder, CO, USA) to pass through an 80-US mesh sieve (0.180 mm). The resulting tortilla flours were stored in plastic bags at - 20 °C until use.
The proximate chemical composition was determined ac- cording to AOAC (2005) standards. Moisture was obtained by drying the samples at 105 °C for 24 h. Protein content was determined using the micro-Kjeldahl method (Nx6.25). Lipid content was determined using a Soxhlet apparatus, with petroleum ether as solvent, and ash was determined incinerating the samples at 550 °C.
Determination of free and bound phenolic compounds Extractions were performed following the protocol by Mora-Rochín et al. (2010). In summary, the free fraction was obtained from 1 g of flour. The free fraction was added to 10 mL of a cold ethanol-water mixture (80:20, v/v), stirred for 10
min and subsequently centrifuged at 2500 x g for 10 min. The supernatant was concentrated using a rotary evaporator at 45 °C and low pressures. The bound phenolic fraction was obtained from the residue of the initial extraction, where 10 mL of NaOH (2 mol/L) were added in a water bath at 95 °C for 30 min, followed by stirring at room temperature for 1 h. The mixture was acidified with concentrated HCl, and stirred for 30 min. Hexane was added for lipid removal. The residue was subjected to 5 extractions with ethyl acetate, evaporated at 45 °C, and finally reconstituted with 50 % methanol. The free and bound phenolic extracts were stored at -20 °C. The phenolic compound content of the different fractions was quantified by the Folin-Ciocalteu method described by Sin- gleton et al. (1999). The results were expressed in milligram gallic acid equivalents (GAE) per 100 g dry weight (dw).
ORAC and ABTS assays were performed as described by Re et al. (1999) and Mora-Rochín et al. (2010). For the measurement of this property by the ORAC method, peroxyl radicals were generated using the AAPH reagent, and the loss of fluores- cence caused by the free radicals was recorded in a micro- plate reader (Synergy HT microplate multi-detection reader; BioTek Instruments, Inc., Winooski, VT, USA). Excitation and emission absorbance measurements were recorded at 485 and 538 nm, respectively.
The ABTS radical (ABTS+) method was performed by the oxidation of 2 mM ABTS with 2.45 mM potassium persulfate (K2S2O8) solution for 12 h. Absorbance of all samples with the
ABTS+ radical was performed at 734 nm on a multi-detection
microplate reader Synergy HT (BioTek Instruments, Inc., Win- ooski, VT, USA) 6 min after initial mixing. A Trolox standard curve was used as a control in both assays. The antioxidant capacity was reported as µmol Trolox equivalents (TE) per g dry weight (dw).
In vitro fermentation of blue maize tortilla flours by co- lonic microbiota
The in vitro fermentation experiments for the nixtamalized and extruded blue maize tortilla flours were carried out as described by Campos-Vega et al. (2009), with minor modifi- cations. Most of the soluble phenolics in the tortilla samples used for fermentation were removed by a wash treatment with cold ethanol-water (80:20, v/v).
Fresh fecal samples were donated by four healthy in- dividuals (marked with letters A-D) ranging in age from 18 to 28 years, with no previous reports of intestinal diseases or antibiotic treatment for at least 3 months. Fecal samples were preserved in sterile containers within 2 h of collection. Two g of each sample were homogenized with 18 mL of 0.1 mol/L sodium phosphate buffer (pH 7.0). This fecal inoculum was used as a fermentation starter. Sterile tubes (15 mL) were filled with 9 mL of sterile basal culture medium. The tubes were sealed and kept under H2-CO2-N2 conditions (10:10:80,
by volume), free of O2 for 24 h. The tubes were inoculated
with 1 mL of fecal matter and 0.1 g of tortilla flour, except for
the blanks. The samples were shaken for 30 s and placed in a 37 °C water bath. Meanwhile, two different controls were performed under the following conditions: (i) the flour was incubated in buffer solution without feces to determine the possible release of phenols, and (ii) the fecal suspension was incubated without flour as a negative control. The samples and controls were collected at 0, 1, and 5 h. Fermentation was terminated by placing the tubes in a -70 °C freezer. The phe- nolics released by fermentation and the antioxidant activity were analyzed by the aforementioned assays.
The percentage of phenolic compounds (PC) that could be available for uptake following the in vitro fermentation was obtained using the following formula (Blancas-Benítez et al., 2018):
% B = Eq (1)
Where the PC released in colonic fermentation is PC RFC; the PC associated with the soluble fraction is SPC, and the PC of the insoluble fraction is IPC. The quantification of each phenolic fraction was calculated following said methodolo- gy.
Experiments were performed in triplicate, and data were presented as mean ± standard deviation (SD). Comparisons between means were analyzed using Graphpad Prism v8 sta- tistical software. An analysis of variance (ANOVA), followed by a Tukey test, were carried out to evaluate the differences between the means of the measured parameters. A p < 0.05 was considered statistically significant.
The results of the proximate chemical analysis of tortillas made with nixtamalized (NMT) and extruded blue maize flour (EMT) are shown in Table 1. The results show similar moisture values (p > 0.05) among tortillas. However, EMT pre- sented the highest values (p < 0.05) for protein (9.31 ± 0.06), lipids (2.03 ± 0.20), and ash (1.41 ± 0.09), while NMT showed the highest value (p < 0.05) for carbohydrate content (83.34
± 0.08).
The higher protein content in EMT compared to NMT, is due to the protein denaturation and the improvement of protein digestibility caused by the high temperatures and
Table 1. Proximate chemical analysis of nixtamalized and extruded blue maize tortillas.
Tabla 1. Análisis químico proximal de tortillas de maíz azul nixtamalizado y extrudido.
Tortillas | Humidity Proteins Lipids Ash | Carbohydrates |
NMT | 48.0 ± 0.0a 9.0 ± 0.20b 1.38 ± 0.10b 1.36 ± 0.05b | 83.34 ± 0.08a |
EMT | 48.0 ± 0.0a 9.31 ± 0.06a 2.03 ± 0.20a 1.41 ± 0.09a | 82.63 ± 0.10b |
Means ± SD. Consecutive means not sharing the same letter are significantly different (p < 0.05). The results are expressed as percentage (%). NMT= nixtamalized maize tortilla, and EMT= extruded maize tortilla.
shearing forces during the extrusion cooking process, which lead to anti-nutritional factors inactivation (such as phytates, tannins, hemagglutinins, and trypsin inhibitors) and thermal unfolding of proteins, changes that enhance bioavailability of sulfur amino acids, as they increase the surface area acces- sible to enzymatic action (Kamau et al., 2020). On the other hand, during the traditional nixtamalization process to pro- duce tortillas, several physical and chemical transformations occur in the kernels during alkaline cooking and subsequent soaking. This process improves the protein quality, niacin availability, and calcium content of the grain while softening the pericarp of the seed. The increased pH and high cooking temperatures help grains humidify, soften their endosperm, which leads to pericarp release. Therefore, proteins and other components of the maize kernel, such as soluble carbohydrates, dietary fiber, and vitamins, are leached into the cooking liquid, causing their loss by removing the ne- jayote, leaving the nixtamalized maize products nutritionally deficient (Cervantes-Ramirez et al., 2020; Kamau et al., 2020; Hassan et al., 2023).
Gámez-Valdez et al. (2021) evaluated the proximate composition of extruded and nixtamalized blue maize tor- tillas fortified with amaranth. These authors observed that tortillas made with extruded flours showed higher protein (10.08 and 7.83 %) and lipid (6.11 and 2.40 %) contents com- pared to tortillas made with nixtamalized flours. In addition, protein content in maize kernels could be influenced by such factors as endosperm type, kernel hardness, genetics, climat- ic conditions, soil type, and agricultural practices employed (Sánchez-Nuño et al., 2024). Furthermore, plant fertilization with nitrogen, or the abundant presence of this compound in the soil, increases the total protein content in plant and grain (Salinas-Moreno et al., 2017).
On the other hand, it can be observed that EMT has the highest lipid content with respect to the nixtamalized tortilla (2.03 and 1.38 %). The lower lipid content in nixtamalized products is determined in part by different times and tem- peratures used in cooking the maize kernels, as well as losses of fractions such as pericarp and germ (the fraction with the highest lipid content in maize) during soaking and washing of the kernels (Hassan et al., 2023). In contrast, the saponifi- cation of the oils contained in the maize kernel, due to the effect of the alkali used during the nixtamalization process, causes a decrease in lipids (Salinas-Moreno et al., 2017; Es- calante-Aburto et al., 2020).
With regard to ash content, a significant part of the ash present in maize tortilla comes from calcium incorporated during the nixtamalization process, which is accumulated mainly in the pericarp and germ fraction of the nixtamalized grain (Salinas-Moreno et al., 2017). Part of these components, however, are partially lost during the soaking and washing stages of the nixtamal while the maize kernel is processed integrally during extrusion. Lime is added to obtain the same flavor as flour obtained by the traditional nixtamalization (Aguayo-Rojas et al., 2012). In this study, it can be observed that the tortilla made with extruded blue maize flour has the
highest (p < 0.05) ash content (1.41 %) compared to the tor- tilla made with the traditional nixtamalization process (1.36
%).
The carbohydrate values present in this study coincide with those reported by Escalante-Aburto et al. (2020), who mention that the total carbohydrate content of traditionally processed tortillas ranges from 44.08 to 87.56 %. However, these values depend mainly on the type of maize used and the nixtamalization conditions. Total carbohydrate content shows a complex behavior during the transformation of maize kernels to tortilla; it can increase, remain constant, or decrease due to excessive loss of starch, lipids, proteins, and pericarp components during cooking, soaking, and washing (Escalante-Aburto et al., 2020). Several studies have indicated that nixtamalized samples contain higher levels of amylose compared to raw corn starch, which is attributed to the depolymerization of amylopectin during the nixtamalization process. Some authors proposed that the rise in amylose content in pigmented samples from raw corn to nixtamal results from the separation of amylose and anthocyanins. This separation occurs due to the breaking of ester bonds, which are unstable in alkaline conditions (Serna-Saldívar, 2021; Enríquez-Castro et al., 2022).
The blue color of the different varieties of maize is the result of the presence of water-soluble phytochemicals known as anthocyanins, which have a high capacity to eliminate and/or neutralize free radicals and reactive oxygen species, chelate metals, control signaling pathways, and reduce proinflammatory markers. As a result, they reduce the risk of cardiovascular pathologies, cancer, neurodegeneration, among others (Sánchez-Nuño et al., 2024).
Table 2 shows the results of anthocyanins and phenolic compounds content (free, bound and total) of blue maize tortillas, obtained by traditional nixtamalization and an al- ternative technology such as extrusion cooking. The tortilla made with nixtamalized blue maize (NMT) (4.01 ± 0.09 mg CGE/100 g, dw) presented the highest value (p < 0.05) of anthocyanins compared to the tortilla made with extruded blue maize (EMT) (2.28 ± 0.15 mg CGE/100 g, dw). The lower
Table 2. Bioactive compounds present in nixtamalized and extruded blue maize tortillas.
Tabla 2. Compuestos bioactivos presentes en tortilla de maiz azul nixtamalizado y extrudido.
Tortillas | Anthocyanins1 | Phenolic Compounds2 | ||
Free | Bound | Total | ||
NMT | 4.01 ± 0.09a | 32.30 ± 3.45a | 125.8 ± 16.01b | 158.1 ± 5.2c |
(20 %) | (80 %) | |||
EMT | 2.28 ± 0.15b | 24.42 ± 2.89b | 247.0 ± 23.48a | 271.42 ± 3.2a |
(9 %) | (91 %) | |||
Means ± SD. Consecutive means not sharing the same letter are significantly different (p < 0.05). 1 mg CGE/100 g dw; 2 mg GAE/100 g, dw. The values in parentheses represent the contribution of this fraction to the total content. NMT= nixtamalized maize tortilla, and EMT= extruded maize tortilla.
anthocyanin content in EMT can be attributed to the condi- tions used (temperature and screw speed) during extrusion, as well as to the moisture content of the raw material. High temperatures during extrusion lead to glycosylating sugar loss with consequent opening of the anthocyanin ring and production of colorless chalcones (Ruíz-Gutiérrez et al., 2018; Menchaca-Armenta et al., 2023).
It is important to mention that the blue maize was fragmented before being fed to the extruder, which caused an increase in the contact area of the anthocyanins with the high temperatures used during extrusion, causing a greater loss of this secondary metabolite (Aguayo-Rojas et al., 2012; Menchaca-Armenta et al., 2023). In contrast, our results are lower than those reported by Bonilla-Vega et al. (2022) who evaluated the effect of different extrusion conditions on blue maize anthocyanins. The authors observed values ranging from 6.37 (T: 170 °C and SS: 145 rpm) to 26.04 (T: 68 °C and SS: 78 rpm) mg CGE/100 g dw, depending on the temperature and screw speed used. Similarly, our values are lower than those reported by Colín-Chávez et al. (2020), who show an anthocyanin content of 21.80 mg CGE/100 g dw, in nixtamal- ized blue maize tortillas.
A diet rich in phenolic compounds has been linked to the prevention of different diseases (different types of can- cer, neurodegenerative, and cardiovascular diseases). Of the cereals consumed worldwide, maize has the highest content of phenolic compounds, mainly in their insoluble form or bound to cell wall materials. However, maize must be pro- cessed prior to human intake, so it is of vital importance to understand the effects of processing on its nutritional value and bioactive compound content (Butts-Wilmsmeyer et al., 2018).
The content of NMT and EMT bound phenolic com- pounds accounted for 80 and 91 % of the total phenolics present in the tortillas, respectively. These values are in agreement with those reported by Roccheti et al. (2022) who mention that most of the phenolic compounds present in cereals are bound, constituting approximately 85 % of the total phenolics present in maize. In contrast, there are reports that mention that different processes applied to foods can enhance the release of bound phenols, among them are fermentation and malting, as well as thermomechanical pro- cesses such as extrusion cooking (Roccheti et al., 2022). EMT presented the highest content of phenolic compounds in the bound (91 %) and total fraction (247.0 ± 23.48 and 271.42
± 3.2 mg GAE/100 g, respectively) when compared to NMT (125.8 ± 16.01 and 158.1 ± 5.2 mg GAE/100 g, respectively).
The results found in this study agree with those report- ed by Gámez-Valdéz et al. (2022), who in a study with com- mercial blue maize tortillas found values of free and bound phenolic compounds of 61.85 ± 0.58 and 134.59 ± 0.83 mg GAE/100 g, dw, and in extruded blue maize tortillas of 64.18
± 0.37 and 218.43 ± 2.83 mg GAE/100 g, dw, respectively. Similarly, our results are consistent with those reported by Bonilla-Vega et al. (2022) who evaluated 13 extrusion condi- tions in a blue maize variety, observing values of total pheno-
lic compounds from 268.06 to 330.53 mg GAE/100 g, dw. The extrusion conditions of this study, nonetheless, differ from those reported by these authors who observed the highest phenolic compound content (330.53 mg GAE/100 g, dw) at a temperature of 50 °C and a screw speed (SS) of 145 rpm. In contrast, the lowest content (268.06 mg GAE/100 g, dw) was obtained at 152 °C and 78 rpm, leading to the conclusion that lower temperatures and higher SS yield the highest total phenolic content.
During extrusion cooking, the cell wall is disrupted, exposing high molecular weight complex phenolic com- pounds to lower molecular weight compounds, resulting in higher extractability of phenolic compounds. However, during this process, the phenolic compound content is also influenced by the origin of the raw material and numerous variables, such as raw material moisture, screw speed, and extruder configuration, i.e., die and screw size, temperature, and exposure time of the sample inside the extruder (Šárka et al., 2021). Likewise, it is important to mention that during extrusion cooking, the maize kernel is processed integrally, and no loss of anatomical parts and leaching of compounds are generated, leaving the extruded products with a higher content of phenolic compounds than those obtained by tra- ditional nixtamalization, in which partial loss of the pericarp (fraction with more than 80 % of the phenolics present in the maize kernel) occurs, and phenolics sensitive to thermal (≥ 85 ºC) and alkaline (pH ≥ 12) conditions are reduced, respec- tively (Dewanto, 2002; Kamau et al., 2020; Astorga-Gaxiola et al., 2023).
Table 3 and 4 show the values of phenolic compounds re- leased by the colonic microbiota from the different donors and the percentage of bioaccessibility, respectively. Tables 3 and 4 show a similar behavior, in which the longer the interaction time between bacteria (contained in feces) from the colon and tortilla flours made with nixtamalized and extruded blue maize, the higher the content of phenolic compounds and their bioaccessibility. At the initial time (0 h) of the evaluation of phenolic compounds released, values were observed between donors (EMT from 0.01 to 0.07 mg GAE/g, and NMT from 0.01 to 0.08 mg GAE/g), while in the bioaccessibility of phenolics no activity could be detected. Interestingly, donors C and D show the highest release of phenolics. In contrast, low release and bioaccessibility values were observed, which may be due to the fact that the inter- action time was not sufficient for the microbiota contained in the fecal samples to carry out the process of metabolizing the phenolic compounds of the bound or insoluble fraction contained in the different tortillas under study. This behavior is similar to that reported by Blancas-Benítez et al. (2018) who were able to observe a phenolic release of 4 to 6 mg GAE/g after 20 min of colonic microbiota interaction in guava flours. During the 1 h fermentation period of this study, a greater release activity of phenolic compounds by bacteria
Table 3. Phenolic compounds released during in vitro colonic fermentation of nixtamalized and extruded blue maize tortillas.
Tabla 3. Compuestos fenólicos liberados durante la fermentación colónica in vitro de tortillas de maíz azul nixtamalizado y extrudido.
NMT EMT | ||||||
Fermentation time (h) | ||||||
DONORS | 0 | 1 | 5 | 0 | 1 | 5 |
A | 0.01 ± 0.01cxB | 2.56 ± 0.06byB | 10.78 ± 0.27ayC | 0.01 ± 0.01cxB | 3.44 ± 0.12bxA | 13.61 ± 0.25axB |
B | 0.03 ± 0.02cxB | 2.70 ± 0.04bxA | 13.81 ± 0.08axB | 0.02 ± 0.01cxB | 0.67 ± 0.03byD | 4.09 ± 0.06ayD |
C | 0.05 ± 0.04cxA | 1.23 ± 0.05byD | 7.71 ± 0.05axD | 0.07 ± 0.04cxA | 1.61 ± 0.02bxC | 7.57 ± 0.02axC |
D | 0.08 ± 0.03cxA | 2.32 ± 0.02byC | 14.83 ± 0.15axA | 0.05 ± 0.03cxA | 2.62 ± 0.18bxB | 14.38 ± 0.13ayA |
Average | 0.042 | 2.20 | 11.78 | 0.037 | 2.08 | 9.91 |
SD | 0.03 | 0.66 | 3.21 | 0.02 | 1.20 | 4.93 |
Means ± SD at 0, 1 and 5 hours (h) of fermentation. Results are the average of at least 3 independent experiments, expressed as mg GAE/g. Consecutive means not sharing the same letter are significantly different (p < 0.05). a-c Comparison of means between times of the same flour. x-z Comparison of means between the same time of the different tortillas, A-C Comparison of means between donors of each time and tortilla. NMT= nixtamalized maize tortilla, EMT: extruded maize tortilla.
Table 4. Bioaccessibility (%) of phenolic compounds during the in vitro colonic fermentation in nixtamalized and extruded blue maize tortillas.
Tabla 4. Bioaccesibilidad (%) de los compuestos fenólicos durante la fermentación colónica in vitro en tortillas de maíz azul nixtamalizado y extrudido.
NMT | EMT | |||||
Fermentation time (h) | ||||||
DONORS | 0 | 1 | 5 | 0 | 1 | 5 |
A | ND | 59.97 ± 0.01bxB | 87.97 ± 0.02axC | ND | 40.3 ± 0.02byB | 74.96 ± 0.23ayC |
B | ND | 60.45 ± 0.02bxA | 88.83 ± 0.21axB | ND | 20.53 ± 0.01byD | 63.53 ± 0.22ayD |
C | ND | 43.73 ± 0.01bxD | 85.07 ± 0.15axD | ND | 38.4 ± 0.01byC | 76.53 ± 0.12ayB |
D | ND | 59.49 ± 0.03bxC | 91.05 ± 0.05axA | ND | 48.38 ± 0.01byA | 83.87 ± 0.15ayA |
Average | ND | 55.91 | 88.23 | ND | 36.90 | 74.72 |
SD | ND | 8.12 | 2.47 | ND | 11.74 | 8.41 |
Means ± SD at 0, 1 and 5 hours (h) of fermentation. Results are the average (%) of at least 3 independent experiments. Consecutive means not sharing the same letter are significantly different (p < 0.05). a-b Comparison of means between times of the same flour. x-z Comparison of means between the same time of the different tortillas, A-C Comparison of means between donors of each time and tortilla. ND= not detected, NMT= nixtamalized maize tortilla, EMT: extruded maize tortilla.
from the colon microbiota was observed, where EMT showed values from 0.67 to 3.44 mg GAE/g, and NMT from 1.23 to
2.70 mg GAE/g, being donor A in EMT, and donor B in NMT the ones that presented the highest phenolic release. During this fermentation time, bioaccessibility of the phenolics released in the different tortillas under study was observed. Bioaccessibility percentages were higher in NMT with values ranging from 43.73 to 60.45 %, while EMT presented values ranging from 20.53 to 48.38 %.
Finally, at 5 h both tortillas presented the highest pheno- lic release and bioaccessibility (EMT = 7.57 to 14.38 mg GAE/1 g, 63.53 to 83.87 % and NMT = 7.71 to 14.83 mg GAE/1 g =
85.07 to 91.05 %). Based on these results, it was be observed that at 5 h, the average phenolic release (11.79 mg GAE/g) and bioaccessibility (88.23 %) among all donors were higher in NMT. Different authors mention that several factors affect the bioaccessibility of phenolic compounds, such as the type of process and food matrix interactions (Tomás-Barberán and
Espín, 2019; Martini et al., 2021). The highest bioaccessibility values in the tortilla obtained from the nixtamalized grain could be attributed to the characteristics of the process, where the maize grain is subjected to high temperature cooking, alkaline pH (> 12), and long resting times. The alka- line conditions that take place during this process can modify or weaken the cell wall structure (formed by cellulose and lignin polymers) favoring enzymatic hydrolysis by the colonic microbiota, thus facilitating the release of phenolic-bound compounds in the different polymers (cellulose, lignin and hemicellulose) that constitute the cell wall (Ying et al., 2018; Baky et al., 2022)
In cereals, phenolic compounds are covalently bound to cell wall polysaccharides, which results in a very low and limited bioavailability for their bioaccessibility in the organism. These complex polysaccharides cannot be hy- drolyzed by enzymes in the gastrointestinal tract, therefore, complexed phenolics are not released and absorbed at the
intestinal level (Tomás-Barberán and Espín, 2019). However, the colonic microbiota will release and bioconvert phenolic compounds attached to the cell wall, producing compounds of smaller molecular size and microbial metabolites, some of which have been shown to be more bioactive and more easily absorbed than their precursors (Tomás-Barberán and Espín, 2020; Astorga-Gaxiola et al., 2023). Likewise, the trans- formation of phenolic compounds by the colonic microbiota modulates their bioactivity, exerting an anti-inflammatory, antioxidant and anticarcinogenic action (Domínguez-Ávila et al., 2020). On the other hand, several authors have men- tioned that technological and biotechnological processes for food production, can induce chemical or physical modifica- tions in foods, or directly influence polyphenols to improve their bioaccessibility and bioavailability (Ribas-Agustí et al., 2018; Calvo-Lerma et al., 2020). These modifications include changes in the food matrix structure leading to the release of phenolic compounds from the matrix and conversion to post- biotic metabolites by intestinal microbial strains (Polia et al., 2022). However, a more comprehensive research approach is still needed to reach valid conclusions on the effects of food processing and polyphenols on human health, mediated by the colonic microbiota (Tomás-Barberán and Espín, 2019).
Incidentally, the results found in this study show similarity to those observed by Juániz et al. (2017), who evaluated colonic fermentation at different times (15 min, 5 and 24 h) in raw, fried (olive oil and sunflower oil) and roasted cardon. These authors were able to observe how the type of processing influenced the release of phenolic compounds during in vitro fermentation, detecting con- siderable microbial metabolic activity as fermentation time increased, observing maximum phenolic release values at 24 h in cardon fried with sunflower oil (8.814 µmol of phenolic compounds/g, dw) compared to unprocessed cardon (0.714 µmol of phenolic compounds/g, dw). Similarly, our results agree with Inada et al. (2020) who evaluated the bioaccessi- bility of phenolic compounds from jaboticaba peel and seeds after gastrointestinal digestion and in vitro fermentation with colonic microbiota. These authors observed an increase in the release of total phenolic compounds in the first 4 h of fermentation (49 %), followed by a decrease after 24 h (17 %). These authors observed that after gastric and intestinal di- gestion, the overall bioaccessibility of phenolic compounds reached 49 %. Unlike other studies that can be found in the literature, our work did not pool the fecal samples, and we evaluated the fermentation of phenolic compounds and bioaccessibility in 5 donors (A-D) individually, being donor D the one who presented the highest values for both evalua- tions. Several authors have shown that interventions on the microbiota do not have uniform effects in different subjects, but that their outcome depends on the individual’s baseline microbiota, both in vivo and in vitro assays. Thus, by pooling fecal microbiota, interindividual differences are completely eliminated and an “artificial” community with unpredictable competition and balance between taxa is created. In contrast to the recent suggestion that microbiota should be pooled
for pooled fermentations, the use of individual, well-pro- tected, fresh fecal microbiota as inoculum for in vitro human gut microbiota experiments, is recommended to avoid un- predictable tendencies (Hou et al., 2020; Perez-Burillo et al., 2021; Isenring et al., 2023).
In our study, antioxidant activity was evaluated by ORAC and ABTS chemical methods. In the ORAC assay (Figure 1) it was observed that, at the initial time (0 h), the antioxidant activity of phenolics released by the microbiota was similar between the NMT and EMT (p > 0.05). However, after 1 h, significant differences were observed, with EMT presenting the highest antioxidant activity values (A = 137.16 ± 13.39, B = 150.07
± 3.80, C = 71.02 ± 1.36, D = 100.09 ± 6.38 µmol TE/g). In
contrast, at 5 h post-fermentation the highest activity was observed mainly in NMT (A = 676.12 ± 76.45, B = 804.57 ± 23.62, C = 282.81 ± 16.75, D = 515.77 ± 11.22 µmol TE/g).
In the ABTS assay, the antioxidant activity showed a similar behavior. During the initial time (0 h), no significant differences (p > 0.05) were observed between tortillas and individuals. However, this changed after 1 h, where the val- ues were very variable depending on the individual, having a similar behavior among tortillas (Figure 2). Finally, at 5 h the NMT reported, for the most part, the highest (p < 0.05) values (A = 24.73 ± 0.14, B = 24.26 ± 0.195, C = 29.32 ± 0.468, and
D = 28.73 ± 0.49 µmol TE/g) compared to its EMT counter- part (A = 24.99 ± 0.14, B = 20.19 ± 0.28, C = 18.31 ± 0.14, D
= 27.01 ± 0.30). As in the phenolic released during colonic fermentation assay, in the evaluation of antioxidant capacity, different individuals had the highest values along the assays (ORAC and ABTS) for both NMT and EMT. Pérez-Burillo et al. (2021) mention that it is important to consider interindividu- al variability during the metabolism of phenolic compounds, as it may lead to different results (different metabolites and/ or physiological effects). Therefore, what is beneficial for one person may be less positive or even unnecessary for another. In both antioxidant activity assays, it was observed that NMT presented the highest average of all donors results at 5 h (569.82 µmol TE/g, by ORAC, and 26.76 µmol TE/g, by ABTS). Kamau et al. (2020) mention that ferulic acid, the most abun- dant phenolic acid in maize with a high antioxidant potential, is mainly found in the trans esterified form to arabinoxylans and hemicelluloses, so more than 90 % is bound in insoluble form. In traditional nixtamalization, the lime in the soaking water can cleave the ester bound, thus releasing this phe- nolic acid. This phenomenon explains the increase of ferulic acid during nixtamalization as reported by Mora-Rochín et al. (2010), who observed a higher percentage of free ferulic acid in tortillas nixtamalized from white maize when compared to
tortillas obtained from extruded maize.
Natural antioxidants are often varied, so it is sometimes a problem to use a one-dimensional method to evaluate this property. The use of more than one methodology to assess the antioxidant activity of foods is essential as they are com- plementary, and their sensitivity depends on the different
macromolecules and bioactive compounds present in the food (Bello-Pérez et al., 2015; Chen et al., 2022). Our results agree with Aguayo-Rojas et al. (2012) who reported that total phenolic compounds are the most important contributors to antioxidant capacity, and that hydrophilic antioxidant activity accounts for approximately 98 % of total antioxidant activity in maize. Additionally, our results contrast with Gax- iola-Cuevas et al. (2017) who evaluated total phenolics and antioxidant activity of different creole blue maize processed by extrusion and traditional nixtamalization. These authors reported that, in both cooking procedures, antioxidant activity decreased compared to raw kernels. However, in their study tortillas made with extruded blue maize retained a higher proportion of total phenolics (83.5 ± 2.1 %) and cellular antioxidant activity (77.5 ± 2.5 %), compared to traditional tortillas (49.8 ± 1.6 and 48.7 ± 1.5 %). In addition, phenolic compounds interact with macromolecules such as proteins, carbohydrates, and lipids, significantly influencing the nutritional and nutraceutical properties of food (Sęczyk et al., 2021b). These interactions are governed by various factors, including external conditions like temperature, pH, and ionic strength, as well as intrinsic properties such as the type, structure, and concentration of both the phenolic compounds and the food matrix components. Despite their well-recognized antioxidant activity, the binding of pheno- lics to food matrices or their interactions with low molecular weight components, such as other phenolics, vitamins, and minerals, often diminishes this activity (Shahidi et al., 2018; Sęczyk et al., 2021a; Sęczyk et al., 2021b).
The composition and physicochemical properties of food matrices, along with processing methods, play a crucial role in modulating the bioaccessibility and antioxidant activi- ty of phenolic compounds. Innovative processing techniques that enhance the stability of phenolics and optimize food matrix properties, can significantly improve their release and availability during digestion (Ribas-Agustí et al., 2018). Moreover, the gut microbiota exerts a substantial influence on polyphenol bioaccessibility, through metabolic conver- sions and enzymatic breakdown of food matrix components. However, since the majority of microbial fermentation occurs in the colon, while absorption predominantly takes place in the stomach and small intestine, the overall bioavailability of polyphenols may be limited (Dufour et al., 2018; Ribas-Agustí
et al., 2018; Cao et al., 2021). Although in vitro models offer valuable preliminary insights into the bioaccessibility of phe- nolic compounds, human studies remain the gold standard for generating accurate and specific conclusions about their behavior and potential health benefits (Sęczyk et al., 2021).
A correlation analysis was performed between the type of processing (extrusion and nixtamalization), antioxidant activity (ORAC and ABTS), bioaccessibility and phenolic compounds released at different fermentation times (Table 5). A strong correlation (R2 = 0.72 - 0.903) (p < 0.05) was ob- served between fermentation time and bioaccessibility (% B), antioxidant activity and phenolic compounds released. In contrast, when evaluating the correlation of treatment (or maize processing) and bioaccessibility, antioxidant activity and phenolic compounds released, the results were negative (R2= -0.029 to -0.156). However, the results obtained show a higher correlation of antioxidant activity and phenolic com- pounds released by the ORAC (R2 = 0.855) than the ABTS (R2
= 0.709) assays. This positive correlation with the ORAC assay may be due to the principle of the assay, which measures the radical chain-breaking capacity of antioxidants by mon- itoring the inhibition of peroxyl radical-induced oxidation, whereas the ABTS assay measures the ability of antioxidants to scavenge free radicals (Cheng et al., 2020; Astorga-Gaxiola et al., 2023).
The positive correlation between time and antioxidant activity in our study agrees with Cheng et al. (2020), who report that the release of phytochemical compounds with antioxidant activity is closely associated with fermentation time. Likewise, these authors observed a positive correlation between total phenolic compounds and antioxidant activity (DPPH and ABTS) after fermentation with Lactobacillus casei, showing that fermentation can increase the antioxidant phenolic compounds involved in the DPPH reduction reac- tion (R2 = 0.998) through proton donation, while the positive correlation with the ABTS assay (R2 = 0.959) may be due to the hydroxyl and phenolic groups that converted ABTS-+ to ABTS. Bello-Pérez et al. (2015) evaluated the correlation between the content of phenolic compounds and tannins
Table 5. Correlation matrix of the variables involved in colonic fermentation in nixtamalized and extruded blue maize tortillas.
Tabla 5. Matriz de correlación de las variables involucradas en la fermentación colónica en tortillas de maíz azul nixtamalizado y extrudido.
Time | Phenolics | Bioaccesibility | ORAC | ABTS | Treatment | |
Time | 1 | |||||
Phenolics | 0.9032175 | 1 | ||||
Bioaccesibility | 0.884294 | 0.84653016 | 1 | |||
ORAC | 0.90266572 | 0.85507923 | 0.78798375 | 1 | ||
ABTS | 0.7174433 | 0.70915823 | 0.90049208 | 0.63874146 | 1 | |
Treatment | 0 | -0.05583935 | -0.15683367 | -0.02977879 | -0.09059565 | 1 |
with two different antioxidant activity methods (DPPH and FRAP). Pearson’s coefficients indicated positive correlations between extractable phenolic compounds and DPPH inhibi- tion (R2 = 0.93), and between condensed tannins with FRAP (R2 = 0.62), suggesting a direct relationship between phenolic compound content and antioxidant activity. The differences in Pearson’s coefficients between the antioxidant methods used in this study, are due to the polarity of the compounds and the sensitivity characteristics of each technique.
The results of the present research highlight that, while both traditional nixtamalization and extrusion processes provide substantial benefits for releasing bioactive compounds, extrusion proves more effective in releasing bound phenolic compounds and enhancing total antioxidant activity. This makes extrusion a promising technology for maximizing the nutraceutical potential of maize-based foods. Notably, tra- ditional nixtamalization remains valued today for its ability to preserve various bioactive compounds with antioxidant benefits that contribute to health protection. This ancestral process, commonly used in tortilla production, preserves the cultural and sensory properties of maize. Additionally, it pro- duces highly bioavailable phenolic compounds after colonic fermentation, which could play a crucial role in the digestive health and prevention of chronic diseases. For this reason, traditional nixtamalization remains the gold standard in tor- tilla production, preserving not only the nutritional quality and nutraceuticals, but also the culinary heritage.
The present work was financially supported by grants from Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCyT-México; grant No. 168279 and 263352).
The author declare no conflicts of interest.
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