Nutritional Composition, Phenolic Compounds, and Antioxidant Capacity of Blue Corn Tortillas Fortified with Quelites (Amaranthus hybridus L.)

Abstract

Tortillas are an essential food staple in the Mexican diet due to their nutritional value. Blue corn tortillas have been reported as a source of bioactive compounds, such as phenolic compounds and flavonoids. Likewise, the blue corn tortillas have been studied to enhance the nutritional and nutraceutical composition. In this sense, Quelites are a large family of plants with macronutrient and micronutrient content, as well as a source of phenolic compounds, flavonoids, and carotenoids. Among these, Amaranthus hybridus L. could fortify the blue corn tortilla composition. Therefore, this study aims to fortify blue corn tortillas with different concentrations of Quelites flours. The total flavonoid and phenolic compounds content, as well as the antioxidant capacity and identification of phenolic compounds, were evaluated on tortillas fortified with Quelites. The addition of Quelites to blue corn tortillas reduced the lipid and protein content, carbohydrate, and flavonoid content, and enhance the antioxidant capacity of tortillas as measured by FRAP, ORAC, and TEAC assays. Also, caffeic acid, chlorogenic acid, ferulic acid, and sinapic acid were identified on blue corn tortillas fortified with Quelites. These results support the use of A. hybridus L. as an ingredient to improve the nutrient and nutraceutical composition of foods.

1. Introduction

In the last decades, the challenge of securing global food and nutrition has called for the urgent exploration and revalorization of underutilized traditional food systems. Particularly, those exhibiting high resilience and dense nutritional profiles. In this context, Quelites, a polyphyletic group of edible non-crop plants deeply rooted in Mexican heritage, represent a crucial advantage for promoting sustainable diets and combating the dual burden of malnutrition [1,2].

Quelites encompass over 500 species whose leaves, stems, and flowers have sustained indigenous and rural communities since pre-Hispanic times, often flourishing in marginal lands or as co-cultivars in the milpa agroecosystem [2,3]. Despite their historical significance and demonstrated adaptability, the consumption and knowledge of Quelites have diminished in recent decades due to shifting dietary preferences and agricultural modernization [1].

Nutritionally, Quelites provide substantial amounts of essential macronutrients and micronutrients, notably high fiber and vegetable protein content, with some species exhibiting protein levels exceeding 22% of dry matter, along with crucial minerals like iron, calcium, and zinc [4,5,6,7]. Furthermore, these plants are potent sources of bioactive compounds, including flavonoids (quercetin and kaempferol derivatives), phenolic acids (caffeic and ferulic acid), and carotenoids, which confer not only remarkable antioxidant capacity [5,6,7,8] but also functional benefits like anti-inflammatory, anti-hyperlipidemic, and anti-diabetic activities, often mediated through the inhibition of key metabolic enzymes such as α-glucosidase and pancreatic lipase, making them ideal candidates for dietary interventions against chronic diseases [3,7,9].

Among these, Amaranthus hybridus L. has been reported to be a rich source of protein, inorganic nutrients, carotenoids, phenolic compounds, flavonoids, and ascorbic acid. Additionally, these Quelites are a source of dietary fiber, calcium, potassium, and magnesium. Also, A. hybridus L. exhibits antioxidant capacity, suggesting that it could be an economically accessible food with potential benefits for consumers [6,10,11].

In this context, Quelites can be used as functional ingredients, serving as one strategy for their incorporation into corn tortillas, a staple of the Mexican diet. It has also been recognized as intangible cultural heritage by UNESCO and is among the most consumed foods in Latin American cuisine [12,13]. Additionally, tortillas are an important contributor to nutrient intake, making them an ideal vehicle for fortification [14]. Also, white maize has been used for tortilla production by traditional processes, namely nixtamalization [10,15], and pigmented maize, such as blue maize, has been studied [16,17]. Studies on fortifying corn tortillas with other traditional legumes, such as common bean flour [18], cabbage [19], groundnut flour [20], among other sources have already demonstrated that this approach successfully improves protein, dietary fiber, phenolic, and total flavonoid content while maintaining consumer acceptability, physical quality, as well as confers potential bioactivity [10]. Therefore, the objective of this work is to characterize the phytochemical profile, antioxidant capacity, and proximate composition of fortified blue corn tortillas with Quelites flour (Amaranthus hybridus L.).

2. Materials and Methods

2.1. Plant Material

Raw Quelites (Amaranthus hybridus L.) and blue corn flour used in this study were purchased in markets located in Culiacán, Sinaloa, Mexico. Blue corn flour packages were stored at environmental temperature, and raw Quelites (A. hybridus L.) were blanched and stored in plastic bags at 4 °C until their use.

2.2. Chemical Reagents

2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) (Sigma 10102946001), 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH) (Sigma 440914), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) (Sigma 238813), fluorescein (Sigma 46955), 2,4,6-Tri-(2-pyridyl)-s-triazine (TPTZ) (Sigma T1253), iron(III) chloride hexahydrate (FeCl3·6H2O) (Sigma 236489), and analytical-grade reagents were also purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.3. Quelites (A. hybridus L.) Blanching Procedure

Blanching was performed as described by Maila and Tseke [21] with slight modifications. Briefly, Quelites (A. hybridus L.) were chopped into approximately 2–3 cm pieces to facilitate blanching, and the leaves and tender stems with damaged or lignified parts were discarded. All chopped pieces were blanched in boiling water (at a 1:5 w/v Quelites–water ratio) for 2–3 min. Immediately afterward, a thermal shock was performed by immersing them in ice water to halt cooking and preserve color and nutrients. Finally, the blanched Quelites (A. hybridus L.) were drained and stored in resealable plastic bags in a freezer to use the next day.

2.4. Production of Corn Tortillas Fortified with Quelites (Amaranthus hybridus L.)

The production of the fortified tortillas followed the methodology described by Astorga-Gaxiola et al. [14], with minor modifications. Blanched Quelites were used as the starting material. These were processed in an industrial blender or food processor, with a minimal addition of potable water (if necessary for grinding) until a fine, homogeneous paste was obtained. This paste was incorporated at concentrations of 10%, 20%, and 30% by wet weight into the blue maize dough. Constant manual kneading was performed until a homogenous distribution was achieved. The dough’s moisture content was adjusted as needed. Once the dough was obtained, small portions (30 g) were used to obtain disks (15 cm) using a manual press (Casa Herrera, México City, México). The disks were cooked to obtain tortillas, which 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 flour was stored in plastic bags at 4 °C until use.

2.5. Proximate Chemical Composition

Moisture, ash, protein, lipids, and carbohydrate contents were determined by the official methods 925.098, 942.05, 920.39, and 32-05.01 of the AOAC, respectively [22].

2.6. Obtention of the Extracts of Samples

Extracts from tortilla flour were obtained according to the method described by Picos-Salas et al. [22]. In brief, 1 g of flour was added to 10 mL of a methanol–water mixture (80:20, w/v), and the mixture was incubated in the dark at constant shaking for 2 h. Later, the mixture was centrifuged at 11,627× g for 14 min at 4 °C using a Z 36 HK centrifuge (HERMLE, Gosheim, Germany). Then, the supernatant was collected and stored at −20 °C until further analysis.

2.7. Total Phenolic Content

The total phenolic content (TPC) was determined according to the methodology reported by Picos-Salas et al. [23]. In total, 10 µL of the extract was mixed with 230 µL of distilled water and 10 µL of the Folin–Ciocalteu reagent in a 96-well microplate. After 3 min of incubation, 25 µL of 4 N Na₂CO₃ was added, and the mixture was incubated in the dark for 2 h. Then, absorbance was measured at 725 nm using a Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). Results were expressed as mg of gallic acid equivalents per gram (mg GAE/g) (n = 3).

2.8. Total Flavonoid Content

The total flavonoid content (TFC) was determined according to the methodology reported by Gutiérrez-Grijalva et al. [24]. In total, 10 μL of the extract was mixed with 250 μL of distilled water, 10 μL of 10% AlCl₃, and 10 μL of 1 M C₂H₃KO₂ in a 96-well microplate. The mixture was incubated for 30 min, and the absorbance was measured at 415 nm using a 96-well Synergy HT microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The total flavonoid content was quantified against a quercetin standard curve (0–0.4 mg/mL). Results were ultimately reported as mg of quercetin equivalents per gram (mg QE/g).

2.9. Antioxidant Capacity Assays

2.9.1. Ferric Reducing Antioxidant Power Assay (FRAP)

This antioxidant assay was performed according to the methodology of Benzie and Strain [25]. In total, 30 μL of the extract and 30 μL Trolox standard curve were mixed with 120 μL of the FRAP reagent (1 mL of 30 mM TPTZ, 1 mL of 60 mM FeCl₃·6H₂O, and 10 mL of acetate buffer) in a 96-well microplate. The mixture was incubated in the dark for 4 min. Absorbance of the reaction was read at 590 nm in a Synergy HT microplate reader (BioTek, Inc., Winooski, VT, USA). The results were expressed as millimoles of Trolox equivalent per g (mmol TE/g).

2.9.2. Oxygen Radical Absorbance Capacity (ORAC)

This assay was performed as described by Huang et al. [26]. In total, 25 μL of the extract was placed in a 96-well microplate with transparent bottom and black walls, and 75 mM phosphate buffer was used as blank, and Trolox as standard. Later, the microplate was placed in the Synergy HT microplate reader (BioTek, Inc., Winooski, VT, USA), and 75 μL of 95.8 μM AAPH (radical generator) and 200 μL of 0.96 μM fluorescein were added to each well. The kinetics of fluorescence loss were measured at 485 nm excitation and 580 nm emission wavelengths at 37 °C for 70 min. The results were expressed as millimoles of Trolox equivalent per g (mmol TE/g).

2.9.3. Trolox Equivalent Antioxidant Capacity (TEAC)

This antioxidant assay was performed as described by Karadag et al. [27]. To obtain the ABTS•+ radical, a solution of 1 mL of 2.6 mM potassium persulfate was mixed with 1 mL of ABTS reagent. The mixture was left in the dark at room temperature for 16 h to complete the reaction. For the assay, 10 μL of the extract was added to 190 μL of the reaction mixture, and the mixture was incubated for 2 h in the dark. Absorbance was measured at 734 nm using a Synergy HT microplate reader (BioTek, Inc., Winooski, VT, USA). The results were expressed as millimoles of Trolox equivalent per g (mmol TE/g).

2.10. Identification and Quantification of Phenolic Acids by UHPLC–PDA

Phenolic acids in the extracts were evaluated according to the methodology described by Jeong et al. [28]. An H-Class UHPLC system (Waters Corporation, Milford, MA, USA) coupled to a photodiode array (PDA) detector was used. Phenolic acids in the extract were separated using a UPLC BEH C18 column (1.7 µm × 2.1 mm × 100 mm) maintained at 40 °C. Gradient elution was carried out using 0.1% formic acid in water (A) and 0.1% acidified acetonitrile (B) at a flow rate of 0.25 mL/min. The following gradient program was applied: 0 min, 98% (A); 20 min, 75% (A); 24 min, 340% (A); 27 min, 10% (A); 28 min, 10% (A); 30 min, 98% (A); and 35 min, 98% (A). Identification and quantification of phenolic acids were performed by comparison with calibration curves constructed from the corresponding standards (ferulic acid and sinapic acid at 254 nm and caffeic acid and chlorogenic acid at 280 nm) at concentrations ranging from 12.5 to 100 µg/mL.

2.11. Statistical Analysis

Total phenolic compounds, total flavonoids compounds, ferric reducing antioxidant power assay, oxygen radical absorbance capacity, and Trolox equivalent antioxidant capacity were analyzed by an analysis of variance (ANOVA) with one factor (Quelites concentration) with three levels (10%, 20%, and 30%), and each analysis was performed by triplicate. Also, mean comparisons were evaluated by Tukey’s HSD test using the software Minitab 19 (Minitab LLC, State College, PA, USA). A p-value of <0.05 was considered significant. Data were reported as mean ± standard error of the mean (SEM).

3. Results

3.1. Proximate Chemical Composition of Fortified Tortillas with Quelites

This work evaluated the effect of incorporating Quelites (A. hybridus L.) at varying concentrations (10, 20, and 30%) into blue corn tortillas. The fortification of blue corn tortillas with Quelites significantly modulated the proximate chemical composition (

Table 1. Proximate chemical composition of fortified tortillas with Quelites (Amaranthus hybridus L.).

Samples	Moisture (%)	Ash (%)	Protein (%)	Lipids (%)	Carbohydrates (%)
Blue corn tortilla	6.53 ± 0.02 b	1.68 ± 0.01 e	11.18 ± 0.85 b	3.03 ± 0.09	77.55 ± 0.83 c
Quelites	10.66 ± 0.25 a	17.12 ± 0.19 a	22.00 ± 0.76 a	2.94 ± 0.08 b	47.27 ± 0.56 d
Tortillas with 10% Quelites	5.54 ± 0.06 d	1.93 ± 0.03 d	10.15 ± 0.33 b	2.33 ± 0.06 d	80.04 ± 0.45 b
Tortillas with 20% Quelites	5.17 ± 0.05 e	2.19 ± 0.03 c	9.21 ± 0.50 c	2.17 ± 0.04 e	81.25 ± 0.53 a
Tortillas with 30% Quelites	5.96 ± 0.05 c	2.44 ± 0.05 b	10.33 ± 0.41 b	2.71 ± 0.09 c	78.56 ± 0.47 c

Results are expressed as mean ± standard deviation (n = 3). Different superscript letters within each column are significantly different according to the Tukey test (p ≤ 0.05).

). The Quelites exhibited a marked nutritional advantage over the blue corn tortilla, showing substantially higher protein (22.0 ± 0.76%), ash (17.12 ± 0.19%), and moisture (10.66 ± 0.25%) content than the blue corn tortillas (11.18 ± 0.85%, 1.68 ± 0.01%, and 6.53 ± 0.02%, respectively).

In fortified tortillas with varying concentrations of Quelites (A. hybridus L.), ash content increased. The highest protein content was observed in tortillas with 30% Quelites (10.33 ± 0.41%) and tortillas with 10% Quelites (10.15 ± 0.33%), both of which were similar (p < 0.05) to the blue corn tortilla control (11.18 ± 0.85%). Ash content increased in a concentration-dependent manner, reaching 2.44 ± 0.05% in tortillas with 30% Quelites, a 49% increase relative to the control (blue corn tortilla). In addition, moisture content was inversely related to ash content in the raw materials, with the highest value observed in pure Quelites (10.66 ± 0.25%) and the lowest in tortillas with 20% Quelites (5.17 ± 0.05%).

On the other hand, a beneficial reduction in the lipid fraction was observed across all fortified samples, ranging from 2.17 ± 0.04% to 2.71 ± 0.09%, which was significantly lower than in the control (3.03 ± 0.009%). Conversely, carbohydrate content remained the principal component, and tortillas with 20% Quelites achieved the highest percentage (81.25 ± 0.53%).

3.2. Total Phenolic and Flavonoid Content

The addition of Quelites (A. hybridus L.) increases the phytochemical profile of the tortillas. Specifically, in this work, we evaluated total phenolic (TPC) and total flavonoid (TFC) contents, as shown in

Table 2. Total phenolic and flavonoid contents of fortified tortillas with Quelites.

Samples	Total Phenolic Content (mg GAE/g)	Total Flavonoid Content (mg QE/g)
Blue corn tortilla	0.34 ± 0.02 c	0.09 ± 0.004 d
Quelites	2.05 ± 0.11 a	8.15 ± 0.57 a
Tortillas with 10% Quelites	0.71 ± 0.06 b	0.61 ± 0.05 c
Tortillas with 20% Quelites	0.73 ± 0.04 b	0.70 ± 0.02 c
Tortillas with 30% Quelites	0.83 ± 0.08 b	1.23 ± 0.04 b

Results are expressed as mean ± standard deviation (n = 3). Different superscript letters within each column are significantly different according to the Tukey test (p ≤ 0.05). TPC = Total phenolic content, TFC = Total flavonoid content. Results are expressed as TFC = mg QE/g and TPC = mg GAE/g.

.

The Quelites (A. hybridus L.) demonstrated substantially higher phytochemical concentrations than blue corn tortillas, with TFC (8.15 ± 0.57 mg QE/g) and TPC (2.05 ± 0.11 mg GAE/g). In contrast, blue corn tortillas had TFC (0.09 ± 0.004 mg QE/g) and TPC (0.34 ± 0.02 mg GAE/g). Despite potential losses during cooking, the TFC of the fortified tortillas increased significantly, with the highest concentration observed in tortillas with 30% Quelites (1.23 ± 0.04 mg GAE/g). This represents a more than 40% increase in flavonoid content relative to the unfortified blue corn tortilla control, confirming the important contribution of the Quelites (A. hybridus L.) addition.

On the other hand, all fortified tortilla groups (10, 20, and 30%) exhibited TPC values higher than the blue corn tortillas (control), suggesting the TPC fraction (likely comprising the native blue corn anthocyanins and phenolic compounds from Quelites) was maintained during the cooking processing, or that the concentration by substitution did not offset the inherent richness of the blue corn tortillas (control). Nonetheless, a clear, increasing trend correlated with substitution level was evident among the fortified samples, ranging from 0.71 ± 0.06 mg GAE/g in tortillas with 10% Quelites to 0.83 ± 0.08 mg GAE/g in tortillas with 30% Quelites (p < 0.05).

3.3. Antioxidant Capacity

The functional impact of incorporating Quelites (A. hybridus L.) was evaluated using three antioxidant assays: the Ferric Reducing Antioxidant Power (FRAP) assay, the Oxygen Radical Absorbance Capacity (ORAC) assay, and the TEAC assay for scavenging ABTS radicals.

Table 3. Antioxidant capacity of fortified tortillas with Quelites.

Samples	FRAP (mmol TE/g)	ORAC (mmol TE/g)	TEAC (mmol TE/g)
Blue corn tortillas	1.19 ± 0.08 d	17.18 ± 1.21 c	2.96 ± 0.15 c
Quelites	27.28 ± 2.08 a	88.49 ± 4.61 a	14.14 ± 0.4 a
Tortillas with 10% Quelites	5.05 ± 0.33 c	43.13 ± 0.65 b	5.90 ± 0.02 b
Tortillas with 20% Quelites	5.20 ± 0.29 b,c	45.49 ± 2.47 b	6.27 ± 0.58 b
Tortillas with 30% Quelites	6.46 ± 0.21 b,c	44.01 ± 2.86 b	6.10 ± 0.36 b

Results are expressed as mean ± standard deviation (n = 3). Different superscript letters within each column are significantly different according to the Tukey test (p ≤ 0.05). FRAP = Ferric Reducing Antioxidant Power Assay, ORAC = Oxygen Radical Absorbance Capacity, TEAC = Trolox Equivalent Antioxidant Capacity. Results are expressed as mmol Trolox Equivalent per gram (mmol TE/g).

shows that Quelites (A. hybridus L.) demonstrated significantly higher antioxidant capacity across all three assays than the blue corn tortillas (ORAC: 88.49 ± 4.61 mmol TE/g; FRAP: 27.28 ± 2.08 mmol TE/g; TEAC: 14.14 ± 0.14 mmol TE/g), confirming its high potential as a functional ingredient. The ORAC and FRAP assays showed the highest antioxidant capacity retention in the fortified products. For FRAP, values increased consistently with fortification, culminating in tortillas with 30% (6.46 ± 0.21 mmol TE/g), which reached parity with the control group (1.19 ± 0.08 mmol TE/g), suggesting that Quelites (A. hybridus L.) compounds effectively preserved ferric-reducing capacity. Similarly, ORAC values were maintained across all fortification percentages, in tortillas with 30% (44.01 ± 2.86 mmol TE/g), demonstrating higher activity comparable to the control (blue corn tortilla: 17.18 ± 1.21 mmol TE/g). Also, the ABTS scavenging capacity increased post-processing and fortification with Quelites (A. hybridus L.). All fortified tortillas clustered significantly higher (p ≤ 0.05) than the blue corn tortillas (control) (2.96 ± 0.15 mmol TE/g), indicating that the specific compounds responsible for ABTS radical neutralization were likely maintained and more accessible during cooking.

3.4. Phenolic Compounds Content by UPLC

The phenolic compound content of blue corn tortillas fortified with Quelites (A. hybridus L.) was evaluated by UPLC, with chlorogenic acid, caffeic acid, sinapic acid, and ferulic acid being identified and quantified (

Table 4. Phenolic compounds content by UPLC of blue corn tortillas fortified with Quelites.

Samples	Caffeic Acid	Chlorogenic Acid	Ferulic Acid	Sinapic Acid
Tortillas with 10% Quelites	Detected	13.26 µg/g	Detected	30,259.52 µg/g
Tortillas with 20% Quelites	Detected	12.78 µg/g	428.27 µg/g	Detected
Tortillas with 30% Quelites	190.51 µg/g	Detected	Detected	29,885.97 µg/g

Detected = compounds found above the limit of detection of the H-Class UHPLC system (Waters Corporation, Milford, MA, USA) coupled to a photodiode array (PDA) detector, but below the standard curve used for the wavelength analyzed.

). In this sense, blue corn tortillas with 10% and 20% of Quelites (A. hybridus L.) presented at 13.26 and 12.78 µg/g of chlorogenic acid, respectively. Also, sinapic acid (30259.52 µg/g) was quantified on blue corn tortillas with 10% Quelites, while 428.27 µg/g of ferulic acid was found on blue corn tortillas with the incorporation of 20% of Quelites. On the other hand, blue corn tortillas fortified with 30% Quelites (A. hybridus L.) contain 190.51 µg/g of caffeic acid and 29,885.97 µg/g of sinapic acid.

4. Discussion

The incorporation of plant ingredients into traditional cereal-based foods has been widely reported to modify their nutritional profiles, phytochemical composition, and functional properties. In maize-based products such as tortillas, the addition of plant materials rich in nutrients, including protein and minerals, among other compounds, increases the bioactivity and quality characteristics [12]. In this sense, the protein content observed in the Quelites (A. hybridus L.) (22.0 ± 0.76%) agrees with Román-Cortés and García-Mateos [9], who reported that traditional vegetables like Portulaca oleracea (verdolaga) and Amaranthus spp. (quintoniles) contain up to 25.9% and 26.2% protein (on a fresh-weight basis, respectively), concentrations substantially higher than those of common cultivated vegetables like spinach (2.86%).

In this context, the inclusion of Quelites (A. hybridus L.) maintained the tortilla’s protein content. Tortillas with 30% Quelites had the highest protein content for the fortified tortillas (10.33 ± 0.41%), without statistical differences to the control (blue corn tortillas). This is critical, as the addition of Quelites (A. hybridus L.) enhances the amino acid profile of the maize staple, which is typically deficient in essential amino acids such as lysine Mateos-Maces et al. [29]. These results are consistent with reports on several Quelites species, including Amaranthus spp. (quintonil) and Chenopodium spp. (Quelites cenizo), which have protein concentrations ranging from 22% to over 26% on a dry weight basis, significantly higher than those of cereals [7,29]. In this context, the addition of Quelites (A. hybridus L.) at 10% to 30% (9.21 ± 0.50% to 10.33 ± 0.41) is similar to the control (11.18 ± 0.85), which is comparable to or slightly higher than gains reported by Treviño-Mejía et al. [18] who used traditional legumes for tortilla fortification. For instance, fortifying maize tortillas with common bean flour increased protein from 9.43% to 10.89%, underscoring that Quelites offers a non-legume, leaf-based alternative with comparable protein-boosting potential [6]. On the other hand, the addition of Quelites to blue corn tortillas increases the protein content compared with tortillas obtained by different processes [10]. In this sense, our protein results are higher than those reported for tortillas by Rojo-Gutiérrez et al. [30], and for tortillas obtained by the traditional method (white corn nixtamalization) [31].

On the other hand, the ash content, a key indicator of mineral density, increased by 49% in tortillas with 30% Quelites (2.44 ± 0.05% vs. 1.64 ± 0.13% in the control). This significant gain validates the use of Quelites (A. hybridus L.) as an effective mineral fortifier, offering a natural route to improve micronutrient intake without relying on synthetic mineral salts. Román-Cortés and García-Mateos [9] reported that Quelites such as Portulaca oleracea exhibit high concentrations of essential micronutrients (Fe, Zn, Mg, and Mn), positioning the fortified tortillas as a valuable vehicle for addressing widespread micronutrient deficiencies. In addition to nutritional content, a complementary health benefit was observed in the lipid profile. The fortified tortillas consistently showed a significant reduction in total lipid content across all substitution levels (10, 20, and 30%) (Table 1). These results are similar to those reported by Rojo-Gutiérrez et al. [30]. This is beneficial, as it lowers the caloric density and saturated fat contribution of the maize staple, offering a product that aligns with modern nutritional recommendations aimed at mitigating the risks associated with excessive fat intake and obesity [28].

In maize-based products such as tortillas, the addition of plant materials rich in phenolic compounds may contribute to enhancing the bioactive compounds (flavonoids and phenolic acids) content of food products while interacting with the endogenous phenolics of maize. These interactions can alter both the qualitative composition and the extractability of phytochemicals in the food matrix. Previous studies have demonstrated that fortifying tortillas with plant ingredients, such as legumes, leafy vegetables, or vegetable by-products, significantly increases the total phenolic content and antioxidant capacity of the final product by contributing additional bioactive compounds and potentially synergistic interactions between plant phenolics and maize anthocyanins [10,17].

The raw Quelites (A. hybridus L.) exhibited a Total Flavonoid Content (TFC) of 8.15 ± 0.57 μmol TE/g, which provides phytochemicals (Table 2). This successfully translated into a significant 43% enhancement in TFC in tortillas with 30% Quelites (A. hybridus L.) (1.23 ± 0.04 μmol TE/g), confirming that Quelites is a powerful source of flavonoids, leveraging the concentration of beneficial phytochemicals like phlorizin, a major compound often found in Quelites like Chenopodium berlandieri [6], known for its anti-inflammatory and anti-diabetic properties [28]. However, the decrease in Total Phenolic Content (TPC) across all fortified groups relative to the blue corn control suggests a pronounced sensitivity to the cooking process. This TPC loss corroborates findings from various food-processing studies [6,10,18,32]. Mateos-Maces et al. [29] reported that high heat during tortilla elaboration could cause thermal degradation of polyphenols. This contrasts sharply with the raw Quelites (A. hybridus L.), which itself showed a significantly higher TPC (2.05 ± 0.11 μmol TE/g) than the tortillas, suggesting that the high TPC of the raw Quelites (A. hybridus L.) includes compounds that are highly susceptible to the tortilla-making process. On the other hand, the addition of Quelites to blue corn tortillas increases TPC and TFC content relative to tortillas produced by the traditional method of white corn nixtamalization [17,31] and by blue corn nixtamalization [17]. Also, these results are higher than those reported for tortillas fortified with Ayocote and Quintonil by Rojo-Gutiérrez et al. [30]. In addition, the observed differences in TFC and TPC contents may be attributable to the chemical structures of the flavonoids, which are often more stable as glycosides. In contrast, phenolic acids have been reported to be more thermosensitive during tortilla processing or to denature anthocyanins [9].

The high retention of FRAP and ORAC activities in fortified tortillas with 30% Quelites (A. hybridus L.) indicates that Quelites compounds survive the cooking process and remain highly effective. In this sense, Román-Cortés and García-Mateos [9] found to have the highest antioxidant activity in Chenopodium spp. (huauzontles), correlating strongly with their content of phenolic compounds and flavonoids. The successful transfer of this functional capacity supports the potential of Quelites to confer health benefits, as evidenced by their ability to neutralize free radicals (ORAC) and reduce metal ions (FRAP) [6]. Also, these results are consistent with the report by Méndez-Lagunas et al. [17], which stated that corn tortillas have antioxidant capacity regardless of the tortilla-making process. The low ABTS assay values in the fortified tortillas further highlight the matrix’s complexity. This suggests that thermal processing altered the antioxidant profile, affecting the compounds reactive toward this specific radical, a phenomenon commonly observed in heat-treated plant products [29]. On the other hand, these results are in agreement with those of tortillas fortified with Ayocote and Quintonil reported by Rojo-Gutiérrez et al. [30]. Likewise, these results are higher than reported by Simić et al. [33] for blue corn tortillas. Therefore, the addition of Quelites to blue corn tortillas increases the antioxidant capacity studied by FRAP, ORAC, and TEAC assays

On the other hand, the chromatographic profile of fortified tortillas with Quelites (A. hybridus L.) shows that varying concentrations alter the phenolic composition of tortillas. In this sense, Ćujić Nikolić et al. [34] reported phenolic compound detection in blue corn maize of 117.64 ± 1.6 µg/g and 29.18 ± 0.8 µg/g for caffeic acid and ferulic acid, respectively. Also, Urias-Peraldí et al. [35] reported a chromatographic detection of ferulic acid in different blue maize between 7.78 and 22.2 µg/g. Likewise, Méndez-Lagunas et al. [17] found a ferulic acid content of 2.48 µg/g in blue corn tortillas, a caffeic acid content of 0.087 µg/g, and a chlorogenic acid content of 0.403 µg/g. These results are lower than those found for fortified blue corn tortillas with Quelites. Therefore, the addition of Quelites flour may enhance the phenolic compounds in blue corn tortillas.

The use of plant-based foods is associated with multiple health-related functional properties, including antioxidants, anti-inflammatory, and metabolic regulatory effects, which may contribute to the prevention of chronic diseases, such as cardiovascular diseases, obesity, and type 2 diabetes [12,29]. Therefore, the use of traditional leafy plants, such as A. hybridus, represents a promising strategy to enhance the functional value of traditional foods in Mexican tortillas.

5. Conclusions

Fortification of tortillas with Quelites (A. hybridus L.) improved nutritional quality in a dose-dependent manner. In this sense, tortillas with Quelites at 10% and 30% significantly maintained protein levels compared to the control, as well as a higher ash content, indicating an enhanced mineral density. On the other hand, tortillas with Quelites reduced lipid levels, whereas carbohydrates remained the predominant nutrient. Additionally, bioactive compounds (such as flavonoids) increased substantially, reflecting their stability during cooking. Likewise, the antioxidant capacity of tortillas with Quelites (A. hybridus L.) was partially preserved in the FRAP, ORAC, and TEAC assays. Overall, the incorporation of Quelites (A. hybridus L.) successfully enhanced the nutritional and functional profile of blue corn tortillas without compromising their basic composition. These results support the use of Quelites (A. hybridus L.) as culturally rooted, sustainable fortifying agents to improve the nutrient density of staple foods. Future studies should address sensory acceptability, flavonoid identification and quantification, bioaccessibility, species-specific effects, and tortilla optimization for broader food applications.