Probiotic Potential of Some Lactic Acid Bacteria Isolated from Blue Maize Atole Agrio from Veracruz, México

Abstract

Mexican culture offers a great variety of traditional maize-based fermented foods that are beneficial for human health. Atole agrio (sour atole), prepared from blue maize (Zea mays) in the state of Veracruz, has been scarcely studied as a potential functional food. The purpose of this study was to select endogenous potentially probiotic lactic acid bacteria (LAB) from freshly fermented blue maize atole agrio. Samples of spontaneously fermented atole agrio were used for the isolation of LAB on MRS agar. The abilities to tolerate acidic pH, bile salts, and sodium chloride, as well as surface hydrophobicity and aggregation capabilities, were used as criteria for probiotic potential. Selected LAB were identified using MALDI-TOF-MS. Finally, safety-related characterizations, such as hemolytic activity and antibiotic susceptibility, were performed. In the initial stages of fermentation, the presence of fungi, yeasts, coliform organisms, and LAB were detected, and in the final fermentation process, where the blue atole agrio reached a pH of 4, 49 isolates of LAB were obtained. Sixteen isolates showed high tolerance to pH 2, and seven of them showed tolerance to 3% bile salts and 4% sodium chloride. The seven isolates were identified as Pediococcus pentosaceus. Although the seven isolates showed low hydrophobicity to hexadecane and chloroform, they had medium autoaggregation and coaggregation with pathogens. The seven isolates showed notable antibacterial properties against Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Escherichia coli, and Listeria monocytogenes, as well as good amylolytic capacity. All the P. pentosaceus strains were non-hemolytic, sensible to clindamycin and resistant to the other 11 antibiotics tested. Only subtle differences were found among the seven isolates, which can be considered potential candidates for probiotics. The freshly fermented blue maize atole agrio can be considered a functional food containing potentially probiotic LAB and the antioxidant phenolic compounds present in blue maize.

1. Introduction

Maize is the primary staple cereal in Eastern and Southern Africa, Mexico, and Central America, serving as the main dietary source of carbohydrates and protein. Mexico is among the world’s leading maize producers and holds the greatest diversity of genetic resources, with approximately 59 native varieties classified by morphological traits. Pigmented varieties—such as violet, red, black, and blue—are cultivated across diverse regions and are valued for their bioactive compounds, particularly antioxidants, which provide health benefits and contribute to disease prevention [1,2].

Maize is used in a wide range of foods, most notably tortillas [3], and in numerous traditional fermented beverages that play significant cultural and economic roles, especially among indigenous communities. Key examples include pozol, chorote, tepache, sendechó, axokot, tesgüino, agua agria, and atole agrio (sour atole) [4,5,6].

Atole refers to a hot maize-based drink, traditionally prepared from cooked and fermented dough made from either plain or lime-treated (nixtamalized) maize. The fermented dough is diluted and boiled to gelatinize starch, producing a thick, viscous beverage consumed hot. Atole agrio is produced in southeastern Mexico (Veracruz, Tabasco, Chiapas) and is also known as xocoatole, jocoatole, xucoatole, shucoatole, atolshuco, or atolxuco. It is consumed for medicinal, ritual, and nutritional purposes and is typically homemade, undergoing spontaneous fermentation by a mixed microbiota of bacteria, yeasts, and fungi, leading to considerable variability in fermentation processes and final products [7].

A notable regional variant, atole agrio “de dobla”, is prepared from white maize harvested before full maturity, which increases its fermentable sugar content. The term “de dobla” refers to the practice of bending maize cobs on the plant prior to harvest to prevent microbial spoilage. Traditionally, the beverage undergoes spontaneous fermentation for 24 h, is boiled before consumption, and is particularly popular in Tabasco, especially near Villahermosa.

The beverage is typically sweetened or flavored with cinnamon or cocoa before serving. Fermentation imparts distinctive sensory attributes and functional properties, while environmental factors such as temperature, rainfall, and humidity influence flavor, aroma, color, and texture [8].

Lactic acid bacteria (LAB) are the dominant microbial group in atole agrio prepared from both mature and “de dobla” white maize. The most prevalent species include Lactococcus lactis, Leuconostoc pseudomesenteroides, Lactiplantibacillus plantarum, Levilactobacillus brevis, Loigolactobacillus coryniformis, Pediococcus pentosaceus, and Weissella confusa. These microorganisms produce vitamins, organic acids, bacteriocins, and enzymes [9,10], enhancing the nutritional, physicochemical, and sensory qualities of the beverage [8]. Key functional contributions include the degradation of anti-nutritional factors (e.g., phytic acid) and the increased availability of soluble fiber, soluble arabinoxylans, free phenolic acids, and bioactive peptides [11].

Growing awareness of probiotic health benefits has spurred interest in traditional fermented foods as sources of probiotic strains for potential commercial use. For atole agrio, few studies have characterized LAB diversity and dynamics, and existing work has focused exclusively on white maize, without evaluating the probiotic potential of isolated bacteria and yeasts. No studies have examined fermented maize beverages produced from blue maize.

The objective of this study was to isolate, identify, and assess the probiotic potential of LAB from fermented, uncooked atole agrio (10–12 h fermentation) prepared from blue maize in Tlapacoyan, Veracruz, Mexico. To our knowledge, this is the first report on atole agrio made from blue maize and on the probiotic potential of its LAB.

2. Materials and Methods

2.1. Plant Material

Blue maize (Zea mays) var. Tuxpeño was obtained from local stores at Tlapacoyan municipality, Veracruz, Mexico (20.01902, −97.14559) where the atole agrio was made. The maize was selected, choosing whole grains free from foreign material contamination (insects, wood, straw, and metal fragments).

2.2. Preparation of Atole Agrio

To prepare the beverage, the blue maize was washed and soaked overnight, then ground in a traditional manual maize and grain grinding mill [Corona^®, Bogotá, Colombia]. The coarse meal was placed in an enameled steel pot with drinking water and washed to remove the pericarp, to obtain a dough that was ground again. This process was repeated three times, and the product filtered using a home steel strainer before being poured into an earthen pot, leaving it near a fireplace to maintain a stable temperature (~37 °C). After 10 h of fermentation, the beverage was placed over direct heat until the boiling point was reached (~96 °C) to finish with the addition of sugar and cinnamon to suit the consumer taste (Figure 1). The uncooked fermented samples were taken and cold stored during each stage of the fermentation process, as well as in the final product.

2.3. pH Variation Measurement

The samples were homogenized, and the pH measured using a digital pH meter (Ketotek, MX-601, Xiamen, China) calibrated with buffer solutions of pH 4 and 6.86. The electrode was immersed in the sample, and the reading was taken in duplicate every hour for a total fermentation time of 10 h [10].

2.4. Microbiological Analysis and Lactic Acid Bacteria (LAB) Isolation

Microbial counts were determined by the plate count method in the atole agrio before and after the fermentation and in the final cooked product (before the addition of sugar and cinnamon). The samples were homogenized with 0.1% peptone water and ten-fold diluted as required. Aerobic mesophilic microbes (Plate Count Agar, BD Bioxon, Cuautitlán Izcalli, Mexico) and coliforms (Violet Red Bile Glucose Agar, BD Bioxon) were incubated at 37 °C for 24 h, LAB (MRS Agar, BD Difco, Detroit, MI, USA) at 37 °C for 24 h, and yeasts and molds (Potato Dextrose Agar, BD Bioxon) at 25 °C for 120 h. No antibiotics were added to the media. The results were expressed in CFU/mL sample. For each sampling stage, 5 to 10 individual colonies were randomly selected from the MRS plates and subcultured on MRS agar for further analysis. Presumptive LAB (Gram-positive and catalase negative bacteria) were purified by successive subculturing on MRS plates [9]. Purified isolates were stored at −20 °C in MRS broth supplemented with 40% (w/v) glycerol.

2.5. Probiotic Potential and Characterization of LAB Isolates from Atole Agrio

2.5.1. Tolerance to Different pH Values

The evaluation of pH tolerance was performed according to the methodology of Adugna and Andualem [12] with some modifications. Isolates were grown individually in MRS broth at 37 °C for 16 h, to obtain a concentration of approximately 10⁷ CFU/mL, and the culture was centrifuged 2 min (3000× g, 4 °C). To simulate the gastric environment, the pellet was washed and centrifuged with 5 mL of saline phosphate buffer (pH 7.2), the recovered cell pellet was re-suspended in MRS broth to have an initial concentration of 10⁶ CFU/mL using a plate count vs. absorbance at 560 nm calibration curve. Aliquots of 10 mL of MRS broth adjusted to different pH values (2, 2.5, and 3) with 1 N HCl were inoculated with 1 mL of the culture and incubated for 2 h at 37 °C. After that, 1 mL was taken from each tube of acid suspension and diluted in 9 mL of peptone water to perform serial dilutions and an aliquot of 100 μL was added to MRS agar and plate cultured. The inoculated plates were placed under anaerobic conditions and incubated at 37 °C for 24 to 48 h. To calculate the percentage of bacteria that survived exposure to acidic conditions (i.e., the percentage of viable LAB), the ratio of the count (CFU) of LAB on MRS agar after exposure to the acidic medium (N₁) divided by the initial count of bacteria at the start of experimentation (N₀) was determined:

$$\mathrm{Survival} \mathrm{rate} (\%) = \left({\displaystyle \frac{\mathrm{l}\mathrm{o}\mathrm{g} \mathrm{C}\mathrm{F}\mathrm{U} {\mathrm{N}}_1}{\mathrm{l}\mathrm{o}\mathrm{g} \mathrm{C}\mathrm{F}\mathrm{U} {\mathrm{N}}_0}}\right)\times 100$$

2.5.2. Resistance to Bile Salts

Isolates that showed a level of acid tolerance [12] were pre-selected, cultured overnight in MRS broth at 37 °C to reach a concentration of 10⁸ CFU/mL (bacterial concentration was analyzed by plate count and turbidimetry) and centrifuged at 3000× g for 10 min. Then, the pellet was washed twice with phosphate buffer solution (PBS), pH 7.2 ± 0.01, and resuspended in 0.5, 1.0, 1.5, and 3% (w/v) of bile (BD Difco™ Oxgall/Ox Bile, Detroit, MI, USA) in MRS broth, respectively, and incubated for 24 h at 37 °C.

Serial dilutions were carried out; a 0.1 μL aliquot of each culture was cultured on MRS agar and incubated at 37 °C for 24–48 h. The experiment was performed in triplicate, and the survival rate of each isolate was calculated. The ratio of the number of LAB colonies counted on MRS agar after exposure to bile salts (N₁) divided by the initial concentration of bacteria at time zero (N₀) and multiplied by 100 was used to calculate the percentage of survivors with the same formula used before for pH tolerance.

2.5.3. Halotolerance of the Isolates

Only the isolates with the highest survival rate in the presence of pH 2 and 3% bile were selected for further analysis. Halotolerance tests were then carried out. This assay is important in the case of atole agrio from Nicaragua, which is prepared with green maize and includes salt and Congo peppers instead of sugar [13]. The methodology proposed by González-Quijano et al. [14] was employed, with minor adjustments. Each isolate was cultivated overnight at 37 °C to inoculate 5 mL tubes containing modified MRS broth with 4, 10, or 16% NaCl. The initial inoculum was 1 × 10⁷ CFU/mL, and growth was measured by optical density (OD₅₆₀) for 24 h using a plate count vs. absorbance at 560 nm calibration curve.

2.5.4. Hydrophobicity of the Cell Wall

The organic solvent affinity test was performed in accordance with the methodology established by Oviedo et al. [15], with minor modifications. The isolates to be analyzed were first subcultured overnight in MRS broth at 37 °C. Then, the culture was subjected to centrifugation (3000× g for 10 min). The resulting pellet was then washed twice with PBS (pH 7.2) to resuspend. Subsequently, 3 mL of the bacterial suspension and 0.75 mL of the solvent (hexadecane and chloroform) were added, respectively. The mixture was then vortexed gently for two minutes and allowed to incubate statically for one hour at 37 °C. Subsequently, the optical density (OD₅₆₀) of the aqueous phase was measured. The percentage of hydrophobicity was determined using the following equation, where A_i represents the initial absorbance and A_f the final absorbance of the aqueous phase.

$$\mathrm{Hydrophobicity} \mathrm{of} \mathrm{the} \mathrm{cell} \mathrm{wall} (\%)={\displaystyle \frac{A_i-A_f}{A_i}}\times 100$$

Surface hydrophobicity can be considered a pre-test for the adhesion capacity of probiotic bacteria to epithelial cells and is one of the significant properties involved in the first contact between bacteria and host cells [16].

2.5.5. Autoaggregation Assay

For this test, the methodologies of Wójcik et al. [17] and Oviedo et al. [15] were followed with some modifications. The selected LAB isolates were incubated in MRS broth overnight, and then the cultures were centrifuged at 3000× g and washed twice with PBS (pH 7.2). The bacterial suspension was adjusted to an OD560 of 0.9 ± 0.05, equivalent to 1 × 10⁸ CFU/mL, and 5 mL of the previously OD-adjusted bacterial suspension was mixed vigorously for 10 s and incubated for 4 h. The percentage of autoaggregation was calculated as shown below; where A_t is the absorbance of the cultures measured from the top of the tubes after 4 h of incubation and A₀ is the initial absorbance.

$$\mathrm{A}\%=\left({\displaystyle \frac{1-A_t}{A_0}}\right)\times 100$$

2.5.6. Coaggregation Test

The ability of LAB isolated from the fermentative process of atole agrio to coaggregate with different Gram-positive (Listeria monocytogenes (ATCC 19115) and Staphylococcus aureus subsp. aureus (ATCC 25923)) and Gram-negative (Salmonella enterica subsp. enterica serovar Typhimurium (ATCC 140228) and Escherichia coli (ATCC 45895)) pathogens was evaluated according to Collado et al. [18] with slight modifications. The pathogen strains were maintained on Nutrient Agar (BD Difco) until used.

The seven previously selected LAB isolates used for the autoaggregation assay, and the pathogen strains were incubated overnight in MRS and nutrient broth, respectively, at 37 °C. Each isolate (LAB) and pathogenic strain was washed twice with PBS (pH 7.2), resuspended in the same solution to an OD of 0.9 ± 0.05, and finally mixed (2 mL each) to measure the initial OD. Next, vigorous shaking was applied for 10 s, and the mixtures were incubated for 2 and 4 h and the absorbance of the cultures was measured from the top of the tubes at each incubation time. All experiments were performed in triplicate; the percentage of coaggregation was determined using the following formula:

$$\%\mathrm{CO}.\mathrm{A}=\left[{\displaystyle \frac{A_{pat}+A_{probio}}{2}}-\left({\displaystyle \frac{A_{mix}}{\frac{A_{pat}+A_{probio}}{2}}}\right)\right]$$

where A_pat and A_probio represent the absorbance of the pathogenic strains and probiotic isolates of the separated bacterial suspensions and A_mix represents the absorbance of the mixed of pathogenic strains and probiotic isolates at two time points, 2 and 4 h, respectively.

2.5.7. Antimicrobial Activity

The antagonistic activity of the selected isolates was evaluated by the well diffusion technique [9,15], pathogenic strains were used: L. monocytogenes (ATCC 19115), S. aureus subsp. aureus (ATCC 25923), S. enterica subsp. enterica serovar Typhimurium (ATCC 140228), and E. coli (ATCC 43895). Pathogenic and isolate cultures were subcultured for 24 h at 37 °C. Each pathogen was inoculated in Petri dishes with nutrient agar and Listeria enrichment agar (for L. monocytogenes culture) by carpet culturing with sterile cotton swap covering the medium uniformly; then, wells with a diameter of 0.85 cm were carefully drilled into each plate using a sterile cork borer and 25 μL of each isolated culture (10⁸ CFU/mL) was deposited for each pathogen performing the assay in triplicate. The plates were incubated at 37 °C for 24 h and the evaluation of antimicrobial activity was conducted by measuring the diameters of the inhibition zones surrounding the wells.

2.5.8. Amylolytic Activity

Due to the nature of the product (starch-containing substrate), the isolates were tested for their ability to metabolize starch. LAB with this capacity have been reported in different tropical amylaceous fermented foods, prepared mainly from cassava and cereals (e.g., maize and sorghum). Their role is important since mono- and disaccharides—such as glucose and maltose and sucrose, which occur naturally in cereals, at a lower concentration than starch—are readily available for lactic acid fermentation. The methodology of Väkeväinen et al. [9] was followed with slight modifications. Seven isolates from the fermentative process of sour atole were cultured in MRS broth and incubated at 37 °C for 18 h. Subsequently, these isolates were inoculated on MRS agar supplemented with 1% soluble starch. After incubation for 48 h at 30 °C followed by 24 h at 4 °C, a 4% v/v iodine solution was sprayed. The formation of a clear halo could be observed around amylolytic LAB colonies.

2.5.9. Safety Testing

Hemolytic Activity

The seven isolates were spread on Columbia agar plates, containing 5% w/v sheep blood, and incubated at 37 °C for 48 h. Subsequently, each plate was examined to evaluate the type of hemolysis presented: β-hemolysis (clear zones around colonies), α-hemolysis (green colored zones around the colonies), or γ-hemolysis (no hemolysis, no clear zones around the colonies) [15].

Antibiotic Susceptibility

The seven isolates were cultured in MRS broth at 37 °C for 24 h. Subsequently, 100 μL portions of the bacterial suspensions, each containing 10⁹ CFU/mL, were introduced onto MRS agar plates at 30 °C. Antimicrobial susceptibility test discs (PT-34 ID Multibac I.D., Multidiscs for Gram-positive bacteria, Investigación Diagnóstica ID, Guadalajara, Mexico) were carefully placed on the surface of the agar [15]. The assessment of antibiotic resistance was performed by measuring the diameters of the inhibition zones [15]. This experimental procedure was replicated three times, and the outcomes were categorized as sensitive (S) or resistant (R) based on the presence or absence of inhibition zones.

2.6. Identification of Isolates Using the System VITEK^® MS

Due to its relatively easy sample preparation in comparison to other methods, matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) is becoming an increasingly popular method for protein-based bacterial identification. Two FDA-approved commercial MALDI-TOF-MS instruments, the Bruker Microflex Biotyper and the BioMerieux VITEK MS, are currently available for bacterial ID using protein profiles. Both support substantial databases for ID of unknown profiles and are accurate to within 92–98%. The method and equipment used in this research included the VITEK^® MS plus system (V2.2/CLI2.0.0, V3.0/CLI3.0.0, bioMérieux S.A., Marcy l’Étoile, France) coupled with RUO and IVD databases provided by the Automated Microbiological Identification Laboratory (AIMA) from the ENCB-IPN. From the probiotic potential tests, seven isolates were selected (the LAB with the highest survival rate in the presence of pH 2 and 3% bile) and cultured by cross-streaking on MRS agar and incubated for 24 h at 37 °C; then, one colony was picked for each isolate previously selected by the probiotic potential tests and as LAB of the fermentative process of the atole agrio. The protocol for bacterial identification was followed in which 1 μL of CHCA Matrix was added in each of the prepared wells of the object holder, allowing for the well to dry completely. Next, the sample was assigned using Vitek MS prep Station (Biomerieux, Marcy-l’Étoile, France); then, the spectra were obtained using Vitek MS Acquisition (Biomerieux, Marcy-l’Étoile, France), and finally the results were acquired using MYLA^® (Biomerieux, Marcy-l’Étoile, France) [19].

2.7. Statistical Analysis

The chemical characterization tests were carried out in triplicate and expressed as the arithmetic mean ± standard error of the mean. Differences among groups were evaluated by one-way analysis of variance (ANOVA) with a significance level of α = 0.05 in all samples using the software R student version 1.4.2.1 (R Core Team, Boston, MA, USA 2021) and the Tukey test also at a α = 0.05 level as a post hoc if necessary.

3. Results and Discussion

3.1. pH Variation and Microbial Counts During Atole Agrio Preparation

During fermentation, pH and temperature variations were monitored to track the progression of the process (

Table 1. Control parameters during the fermentation of atole agrio.

Time (h)	pH	Temperature (°C)
0	6.42	22
1	6.05	24
2	5.99	26
3	5.99	30
4	5.99	32
5	5.99	33
6	5.86	34
7	5.28	35
8	4.39	37
9	4	37
10	4	37

). The traditional fermentation method is carried out in a kitchen, where a fireplace is lit to maintain the temperature above 30 °C. After 6 h, the pH decreased to 5.86, and by the end of fermentation (12 h), it reached 4. The temperature range (22–37 °C) was adequate for proper fermentation. These results are similar to those reported for white maize atole agrio, in which the pH decreased from 7 to 4.7 after 6 h at 30–40 °C [9].

At the end of the 12 h fermentation, the pot was placed over direct heat, causing the starch in the maize grains to swell due to gelatinization (>84 °C) [20]. This step resulted in a thicker beverage with the desired consistency.

The pH decrease is attributed to the microbial consortium present in the beverage, which can produce various metabolites, mainly organic acids such as acetate, lactate, and propionate [7]. This acidification plays an important role in developing sensory characteristics and improving nutritional value by degrading complex compounds such as carbohydrates, proteins, and fiber [6].

The color of blue maize atole agrio depends on the pH-sensitive behavior of anthocyanins present in the cereal, due to the ionic nature of their molecular structure. In acidic conditions, some anthocyanins appear red, shift to a purple hue at neutral pH, and turn blue under basic conditions. The red-colored anthocyanin pigments, predominantly in the form of flavylium cations, are more stable in acidic solutions [21]. In blue maize atole agrio, the initial product (pH 6.42) has a purplish-blue color, whereas the final fermented product develops a reddish hue when the pH reaches 4 (Figure 2). Blue maize anthocyanins have been used as natural pH indicators in smart packaging films [22].

Microbial counts of aerobic mesophiles, coliforms, lactic acid bacteria (LAB), yeasts, and molds were determined using the plate count method before and after fermentation, and in the final cooked product (before the addition of sugar and cinnamon) (

Table 2. Microbial counts (log₁₀ CFU/mL) during the blue maize atole agrio manufacturing process.

Microbial Group	Fermentation Start	End of Fermentation	Cooked Product
Aerobic mesophiles	7.30 ± 3.46	7.08 ± 3.34	n.d.
Coliforms	3.18 ± 0.98	n.d.	n.d.
LAB	7.30 ± 3.42	8.23 ± 3.95	n.d.
Yeasts	6.00 ± 2.62	3.18 ± 1.02	n.d.
Molds	6.18 ± 2.79	2.00 ± 0.32	n.d.

n.d.—not detected.

).

LAB, aerobic mesophiles, and yeasts were the dominant microbial groups at the end of fermentation. In the case of white maize atole agrio [9], although LAB counts were higher at the end of fermentation (1.3 × 10⁹ CFU/mL), coliform counts were also higher (6.3 × 10⁷ CFU/mL). This indicates better sanitary quality in blue maize atole agrio, where the antimicrobial properties of phenolic compounds may play an important role in the fermentation process.

3.2. Isolation of Lactic Acid Bacteria

From the final fermented product, 49 LAB isolates were obtained (Gram-positive and catalase-negative). Among them, 28 were identified as short bacilli, 9 as long bacilli, and 12 as cocci. Väkeväinen et al. [9] reported the isolation of 88 LAB from white maize atole agrio, while Otunba et al. [23] reported 11 LAB isolates from fermented sorghum products, including bacilli and cocci. Similarly, Cizeikiene et al. [24] reported the isolation of 10 LAB from spontaneous Lithuanian rye sourdoughs.

3.3. Probiotic Potential Determination of the LAB Isolates

3.3.1. Tolerance to Different pH Values

Of the 49 isolates tested (100%), all were subjected to the pH resistance assay for 2 h. Forty-four isolates (89.8%) survived at pH 3, thirty-nine isolates (79.6%) at pH 2.5, and only sixteen isolates (32.6%) remained viable at pH 2, all with survival rates above 80%. These results led to the selection of the 16 isolates with the highest tolerance to acidic conditions (Table 3), where coccus morphology predominated, showing greater resistance to pH changes.

In the case of white maize atole agrio, strains of Lc. lactis, Lp. plantarum, and P. pentosaceus demonstrated the highest low-pH tolerance [9]. In contrast, Adugna and Andualem [12] found that, among 80 isolates exposed to low pH, only 6 Lactobacillus isolates showed good tolerance.

LAB genera such as Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, and Pediococcus are generally acidophilic, tolerating low pH through various mechanisms, including neutralization reactions, biofilm development, proton pumps, macromolecule shielding, preadaptation and cross-protection, and solute activity [25]. Tolerance to low pH is essential for a LAB strain to be considered as part of a starter culture for the fermentation of foods such as milk, vegetables, and cereals, in which they produce organic acids (lactic, acetic, propionic, and butyric acids) [26]. An indispensable probiotic requirement is the ability to survive human gastric conditions (pH 1.5–3.5) for 30 min to 2 h [27].

Table 3. Survival rate (%) of the LAB isolates with better tolerance to acidic pH after an incubation period of 2 h.

Isolate Code	Morphology	pH 2	pH 2.5	pH 3
AG13	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG19	Long bacillus	81.4 ± 1.7	100 ± 0.0	100 ± 0.0
AG22	Short bacillus	92.5 ± 1.8	100 ± 0.0	100 ± 0.0
AG23	Short bacillus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG24	Short bacillus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG25	Short bacillus	95 ± 0.5	100 ± 0.0	100 ± 0.0
AG 26	Short bacillus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG 27	Short bacillus	81 ± 1.7	95 ± 1.5	100 ± 0.0
AG 29	Coccus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG 34	Coccus	95 ± 1.5	100 ± 0.0	100 ± 0.0
AG 35	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG 39	Coccus	95 ± 1.8	100 ± 0.0	100 ± 0.0
AG 40	Coccus	95 ± 1.7	95 ± 1.5	100 ± 0.0
AG 41	Coccus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG 42	Short bacillus	94 ± 1.5	100 ± 0.0	100 ± 0.0
AG 43	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0

Table 3. Survival rate (%) of the LAB isolates with better tolerance to acidic pH after an incubation period of 2 h.

Isolate Code	Morphology	pH 2	pH 2.5	pH 3
AG13	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG19	Long bacillus	81.4 ± 1.7	100 ± 0.0	100 ± 0.0
AG22	Short bacillus	92.5 ± 1.8	100 ± 0.0	100 ± 0.0
AG23	Short bacillus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG24	Short bacillus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG25	Short bacillus	95 ± 0.5	100 ± 0.0	100 ± 0.0
AG 26	Short bacillus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG 27	Short bacillus	81 ± 1.7	95 ± 1.5	100 ± 0.0
AG 29	Coccus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG 34	Coccus	95 ± 1.5	100 ± 0.0	100 ± 0.0
AG 35	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0
AG 39	Coccus	95 ± 1.8	100 ± 0.0	100 ± 0.0
AG 40	Coccus	95 ± 1.7	95 ± 1.5	100 ± 0.0
AG 41	Coccus	90 ± 1.5	100 ± 0.0	100 ± 0.0
AG 42	Short bacillus	94 ± 1.5	100 ± 0.0	100 ± 0.0
AG 43	Coccus	100 ± 0.0	100 ± 0.0	100 ± 0.0

Figure 3 shows the principal component analysis (PCA) used to visualize the phenotypic variability of the 49 LAB isolates according to their resistance to different pH values, incorporating cell morphology. The model explained 99.17% of total variability, with the first principal component (PC1) accounting for 70.25% and the second (PC2) for 28.92%, indicating adequate representation of the variability observed in the initial two dimensions.

In the graphical projection, short bacilli (blue) tend to cluster centrally, suggesting a relatively homogeneous response to pH changes and similar resistance levels across isolates. This morphology has been associated with efficient adaptation to acid stress, possibly due to smaller surface area exposure and higher cell density, which help protect enzymatic systems [25].

Long bacilli (green) showed greater dispersion, including points with atypical or extreme behavior. This variability suggests the presence of strains with divergent responses to pH, likely due to physiological differences such as membrane composition or proton pumping capacity [28]. While long bacilli may have a higher adaptive potential to fluctuating environments, this is not uniform across strains.

Coccoid morphology (red) was less represented and showed limited dispersion, possibly reflecting intermediate or less diverse pH responses. Previous studies suggest that lactic cocci, despite surviving certain conditions, tend to have more limited pH tolerance than bacilli, likely due to less versatile metabolic strategies [29,30].

3.3.2. Bile Salt Tolerance

The 16 pH 2-tolerant isolates were tested with 0.5%, 1%, 1.5%, and 3% ox bile (DifcoTM Oxall, Detroit, MI, USA) for 24 h at 37 °C. Most isolates tolerated 0.5–1.5% bile with final counts ~6 Log CFU/mL. Only seven isolates tolerated 3% bile: AG13, AG29, AG34, and AG35 maintained ~6–7 Log CFU/mL, while AG40, AG41, and AG43 decreased >50% compared to controls with 0% bile salts (8 Log CFU/mL) (Figure 4).

Bile salts are biological detergents that emulsify and solubilize lipids, playing a crucial role in fat digestion while also exhibiting antibacterial activity, mainly by disrupting bacterial membranes [12]. Conjugated bile salts can cause bacterial toxicity through mechanisms similar to organic acids, primarily by acidifying the cytoplasm [31]. One resistance mechanism involves the hydrolysis of conjugated bile salts, with proton cotransport of the deconjugated product out of the cell; this bile salt hydrolase activity is recognized as an important adaptation strategy [32].

Gil-Rodríguez and Beresford [32] evaluated the growth of various LAB species and found that isolates of L. acidophilus, Lv. brevis, Ls. casei, Lt. curvatus, Ls. paracasei, Lp. plantarum, Ls. rhamnosus, Lg. salivarius, Pediococcus acidilactici, and P. pentosaceus were able to grow at low bile concentrations (0.3%), whereas L. helveticus, Leuconostoc lactis, and L. delbrueckii ssp. bulgaricus were inhibited. This suggests that LAB isolates from blue maize atole agrio fermentation may possess a cholesterol-lowering probiotic potential due to the presence of bile salt hydrolase activity [32].

3.3.3. Identification of Isolates Using the Vitek MS Plus System

The seven isolates showing the best pH and bile salt tolerance were identified using the Vitek^® MS Plus MALDI-TOF system (Biomerieux, Marcy-l’Étoile, France). The results revealed that all of them belong to Pediococcus pentosaceus.

Mass spectrometry has become an important tool for the microbiologist and the biotechnologist for bacterial identification, since matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry (MALDI-TOF-MS) has been revealed to provide reliable protein profiling data to identify many bacteria [17], including LAB [33].

3.3.4. Halotolerance of LAB Isolated from the Fermentative Process of Atole Agrio

The seven P. pentosaceus isolates previously selected for their resistance to pH 2 and 3% bile salt concentration were evaluated for halotolerance. The seven isolates had an initial optical density between 0.15 and 0.2 so all isolates had notably reduced growth in the presence of increasing concentrations of sodium chloride (Figure 5). However, all isolates still exhibited significant growth around 7.5% NaCl, confirming their halotolerant character. The maximum growth of the P. pentosaceus isolates at different concentrations of NaCl and their behavior can be described as a decreasing logistic curve, as previously reported by González-Quijano et al. [14]. All the isolates had similar final bacterial concentrations, and their range of tolerance was from 4 to 10% sodium chloride, still showing weak growth at 16% salt.

Halotolerant microorganisms can be defined as those capable of growing in the absence as well as in the presence of relatively high NaCl concentrations. If growth spreads above 2.5 M (14.61%), they are known as extremely halotolerant [34]. The above results indicate that the seven P. pentosaceus isolates could then be considered as moderate halotolerant microorganisms. The survival rate (%) under different stress conditions of P. pentosaceus isolates from the fermentation process of blue maize atole agrio are shown on Table S1 in Supplementary Material.

3.3.5. Hydrophobicity of the Cell Wall

Figure 6 and Figure 7 show the results for the microbial adhesion to hydrocarbons (MATH) test for measuring the hydrophobicity of the cell wall using hexadecane and toluene. This method is related to the role of hydrophobic interactions in microbial adhesion. The seven selected P. pentosaceus isolates were evaluated and isolates AG13, AG40, and AG43 reached values in hexadecane of 27.6 ± 0.5%, 18.3 ± 0.3%, and 20.7 ± 0.1% after 2 h of incubation. The ability of bacteria to bind to the intestinal epithelium includes several mechanisms, including adhesins present on the surfaces of bacterial cells, which have carbohydrate bonds located in the glycocalyx of enteric cells that function as anchor spots for the bacteria, thus colonizing the intestine and preventing the further adhesion of pathogens [35].

A similar study performed with xylene and hexadecane evidenced hydrophobicity percentages between 25.04 and 35% for Lm. reuteri ATCC 55730, a well-known probiotic that shows good adherence in vivo [29]. These values are in the range of the ones obtained in this study. At present, it appears that hydrophobic surface properties, as measured by MATH, may play a role in predicting adhesion to host cells, aggregation, and flocculation, and that the higher the hydrophobicity, the higher the adhesion to Caco-2 cells and vice versa [16,36].

3.3.6. Autoaggregation

The percentages of autoaggregation (Figure 8) showed values of 24.31% ± 0.8 for AG43 isolate, with the AG29 isolate showing the lowest values of self-aggregation (14.22% ± 0.01). According to García et al. [37], this phenomenon is defined when cells of the same species are aggregated, related to the components of the bacterial wall, so that probiotics or LAB can bind to intestinal cells and stimulate the immune system and act as a barrier against pathogenic microorganisms [38].

The results shown in Figure 8 are similar to those reported by Rahman et al. [39] for bifidobacteria, where the autoaggregation values were divided into high (>70%), medium (20–70%), and low (<20%), so the isolates evaluated in this study are in the range of low and medium autoaggregation values, which are related to the low hydrophobicity presented in Figure 6 and Figure 7. This could be due to the type of P. pentosaceus isolate and also to the possible correlation with the hydrophobicity values [15].

Figure 8. Autoaggregation assay of seven P. pentosaceus strains isolated from the atole agrio fermentation process. Different lowercase letters (a–c) indicate statistically significant differences (p ≤ 0.05).

Figure 8. Autoaggregation assay of seven P. pentosaceus strains isolated from the atole agrio fermentation process. Different lowercase letters (a–c) indicate statistically significant differences (p ≤ 0.05).

3.3.7. Coaggregation

The data showed that the levels of coaggregation between the P. pentosaceus isolates and the tested pathogens (S. aureus, L. monocytogenes, E. coli, and S. enterica) varied, ranging from 20 (AG35) to 28.6% (AG34) after 4 h of incubation. In general, all seven P. pentosaceus isolates coaggregated similarly with the four pathogenic isolates tested.

The ability of LAB to coaggregate with pathogens may explain one of the mechanisms by which they are considered a host defense against infection [15]. Therefore, this assay is considered important to evaluate probiotic potential, since it is involved in the colonization of the gut by microorganisms beneficial to the host [40]. Laurencio et al. [35] evaluated the coaggregation of Lactobacillus spp. with cells of E. coli, S. aureus, and Klebsiella spp., finding that all the P. pentosaceus isolates evaluated showed a coaggregation greater than 50%, except LvB-52 against E. coli (18%).

According to the results, all the P. pentosaceus isolates subjected to the coaggregation test have the capacity to bind to pathogenic bacteria (Gram-positive and Gram-negative) although the percentages may depend on the type of strain in addition to the incubation time, which agrees with the findings provided by Collado et al. [18], who explain that the longer the incubation time, the greater the aggregation. Our results suggest, as in other studies, that the autoaggregation ability of P. pentosaceus isolates may be linked to their capacity to coaggregate with pathogens, as both processes are thought to contribute to the exclusion and inhibition of pathogenic bacteria in the intestinal tract [15,41,42].

3.3.8. Antibacterial and Amylolytic Activities

The seven P. pentosaceus isolates (Table 4) showed notable antibacterial properties against S. aureus, S. enterica, E. coli, and L. monocytogenes. Some photographs of the inhibition zones for the pathogenic strains can be observed in Figure 9. P. pentosaceus isolate AG35 showed the highest antibacterial activity against Sa, AG40 against St, AG41 against Ec, and AG34 against Lm. Sa and Ec showed high susceptibility against the selected isolates, while St and Lm showed medium susceptibility. The bactericidal activities presented in this study were higher than those of the isolates from white maize atole agrio in the state of Tabasco by Väkeväinen et al. [9], which exhibited inhibition halos between 11 and 17 mm maximum. Considering the short fermentation duration of blue maize atole agrio (9 h) and the high bacterial counts with probiotic characteristics, the selected isolates have remarkable antibacterial activities against important pathogens in food safety. The antibacterial activities of P. pentosaceus isolated from plant sources result from the production of primary metabolites, such as organic acids, and mainly lactic acid [43]. Lactic acid has bactericidal activity against pathogens by reducing the proliferation of bacteria susceptible to pH changes through acid stress [44]. Furthermore, some bacteria with probiotic capacity produce secondary metabolites such as bacteriocins or BLIS (bacteriocin-like inhibitory substances), which inhibit the growth and reproduction of numerous bacteria [45]. These metabolites are of great interest in the food sector since they are used in the preservation of food products and human health, highlighting different LAB species in dairy products [46].

Figure 9. Antimicrobial activity of P. pentosaceus strains isolated from blue maize atole agrio. (a) Isolates AG34 and AG35 vs. Staphylococcus aureus. (b) Isolates AG40 and AG41 vs. Escherichia coli. (c) Isolates AG40 and AG41 vs. Salmonella enterica. (d) AG13 and AG29 vs. Listeria monocytogenes.

Figure 9. Antimicrobial activity of P. pentosaceus strains isolated from blue maize atole agrio. (a) Isolates AG34 and AG35 vs. Staphylococcus aureus. (b) Isolates AG40 and AG41 vs. Escherichia coli. (c) Isolates AG40 and AG41 vs. Salmonella enterica. (d) AG13 and AG29 vs. Listeria monocytogenes.

Table 4. Antibacterial and amylolytic action of P. pentosaceus isolates from the fermentation process of blue maize atole agrio.

Isolate Code	Antibacterial Activity (Halo Diameter in mm Measured after 24 h of Incubation at 37 °C)				Amylolytic Activity
	Sa *	St *	Ec *	Lm *
AG13	15.5 ± 0.38	8 ± 0.18	11.5 ± 0.35	12.5 ± 0.31	+
AG29	12 ± 0.23	7.5 ± 0.14	11 ± 0.32	11 ± 0.36	+
AG34	14 ± 0.39	10 ± 0.24	12.5 ± 0.45	14.5 ± 0.42	+
AG35	24.5 ± 0.65	9.5 ± 0.21	15 ± 0.41	13 ± 0.38	+
AG40	10.5 ± 0.29	22.5 ± 0.55	15.5 ± 0.40	14 ± 0.24	+
AG41	12.5 ± 0.33	20 ± 0.36	23.5 ± 0.65	11 ± 0.32	+
AG43	17.5 ± 0.33	11.5 ± 0.38	16.5 ± 0.40	12.6 ± 0.39	+

* Staphylococcus aureus (Sa), Salmonella enterica (St), Escherichia coli (Ec), and Listeria monocytogenes (Lm). + Amylolytic activity is present.

Table 4. Antibacterial and amylolytic action of P. pentosaceus isolates from the fermentation process of blue maize atole agrio.

Isolate Code	Antibacterial Activity (Halo Diameter in mm Measured after 24 h of Incubation at 37 °C)				Amylolytic Activity
	Sa *	St *	Ec *	Lm *
AG13	15.5 ± 0.38	8 ± 0.18	11.5 ± 0.35	12.5 ± 0.31	+
AG29	12 ± 0.23	7.5 ± 0.14	11 ± 0.32	11 ± 0.36	+
AG34	14 ± 0.39	10 ± 0.24	12.5 ± 0.45	14.5 ± 0.42	+
AG35	24.5 ± 0.65	9.5 ± 0.21	15 ± 0.41	13 ± 0.38	+
AG40	10.5 ± 0.29	22.5 ± 0.55	15.5 ± 0.40	14 ± 0.24	+
AG41	12.5 ± 0.33	20 ± 0.36	23.5 ± 0.65	11 ± 0.32	+
AG43	17.5 ± 0.33	11.5 ± 0.38	16.5 ± 0.40	12.6 ± 0.39	+

* Staphylococcus aureus (Sa), Salmonella enterica (St), Escherichia coli (Ec), and Listeria monocytogenes (Lm). + Amylolytic activity is present.

Similarly, the analyzed P. pentosaceus isolates in this study had, as expected, amylolytic capacity (Table 3). Some LAB produce extracellular amylases capable of hydrolyzing starch, highlighting some isolates of Lp. plantarum and Lm. brevis (>9.23 ± 0.39 U/mL). The amylase produced by these bacteria is capable of degrading starch into dextrin and subsequently into glucose [46,47]. Amylolytic LAB probiotics have an ecological advantage when grown in starchy matrixes, in addition to improving the nutritional, physiochemical, and health properties of them [47].

3.3.9. Safety Testing

Hemolytic Activity

The seven P. pentosaceus isolates did not show a hemolytic circle around their colonies, indicating that they were all γ-hemolytic, confirming that they are safe for human use.

Antibiotic Susceptibility

The results for the susceptibility test to 12 antibiotics for the selected seven P. pentosaceus isolates are shown in

Table 5. Antibiotic susceptibility in MRS agar for the P. pentosaceus isolates. Inhibition zone diameter: ≤14 mm indicate resistance, 15–19 mm intermediate resistance and ≥20 mm susceptible.

Antibiotic	AG13	AG29	AG34	AG35	AG40	AG41	AG43
Aerobic mesophiles	3 ± 0.08	4 ± 0.18	4 ± 0.16	4 ± 0.18	3 ± 0.07	4 ± 0.16	3 ± 0.08
Ampicillin 10 μg	12 ± 0.44	10 ± 0.39	9 ± 0.32	12 ± 0.49	11 ± 0.30	10 ± 0.37	10 ± 0.37
Cephalothin 30 μg	4 ± 0.11	5 ± 0.16	5 ± 0.16	6 ± 0.32	6 ± 0.32	6 ± 0.32	4 ± 0.11
Cefotaxime 30 μg	0	0	0	0	0	0	0
Ciprofloxacin 5 μg	18 ± 0.78	20 ± 0.68	19 ± 0.64	21 ± 0.66	19 ± 0.60	19 ± 0.61	17 ± 0.54
Clindamycin 30 μg	2 ± 0.07	3 ± 0.11	1 ± 0.08	3 ± 0.10	3 ± 0.10	2 ± 0.07	3 ± 0.11
Dicloxacillin 1 μg	11 ± 0.35	12 ± 0.48	11 ± 0.35	11 ± 0.36	11 ± 0.35	11 ± 0.35	11 ± 0.34
Erythromycin 15 μg	2 ± 0.11	3 ± 0.18	2 ± 0.06	1 ± 0.05	1 ± 0.06	1 ± 0.05	2 ± 0.07
Gentamicin 10 μg	10 ± 0.42	12 ± 0.49	11 ± 0.32	12 ± 0.49	11 ± 0.32	10 ± 0.41	11 ± 0.31
Penicillin 10 U	5 ± 0.25	8 ± 0.28	7 ± 0.27	5 ± 0.24	5 ± 0.25	5 ± 0.24	6 ± 0.27
Tetracycline 30 μg	0	0	0	0	0	0	0
Sulfamethoxazole/trimethoprim 23.75/1.25 μg	0	0	0	0	0	0	0
Vancomycin 30 μg	3 ± 0.08	4 ± 0.18	4 ± 0.16		3 ± 0.07	4 ± 0.16	3 ± 0.08

. All the isolates were sensible to clindamycin and resistant to the other 11 antibiotics tested. Clindamycin is a lincosamide antibiotic that has been approved by the US FDA for the treatment of anaerobic, streptococcal, and staphylococcal infections. P. pentosaceus was fully resistant (no inhibition halo) to ciprofloxacin (a fluoroquinolone antibiotic), sulfamethoxazole/trimethoprim (sulfonamide antibiotics), and vancomycin (a glycopeptide antibiotic). This could indicate the presence of several resistance mechanisms in this LAB that are effective against chemically different antibiotics. A previous study [48] investigated the antibiotic susceptibility of 35 strains of P. pentosaceus isolated from various food matrices in a period of 70 years. No antibiotic resistance genes could be detected in the genome assemblies of the 35 P. pentosaceus strains analyzed using NCBI AMRFinderPlus, CARD, ARG-ANNOT, and Resfinder. This indicates that the possibility that the antibiotic resistance could eventually be acquired by pathogenic bacteria through horizontal gene transfer is very low. In this same study, all the isolated strains were resistant to tetracycline and vancomycin. In the case of Korean fermented seafoods, the isolated P. pentosaceus strains were resistant to kanamycin and susceptible to ampicillin, erythromycin, clindamycin, tetracycline, and chloramphenicol [49]. All the reported cases of antibiotic resistance in P. pentosaceus involve intrinsic resistance, which is independent of antibiotic selective pressure and horizontal gene transfer. It is achieved by different mechanisms such as a lack of affinity of the antibiotic for the bacteria, expulsion of the antibiotic by chromosomally encoded efflux pumps, an absence of affinity of the drug for the bacteria, and the occurrence of antibiotic-degrading enzymes [48,50].

3.3.10. Pediococcus pentosaceus

Pérez-Cataluña et al. [10] mentioned that isolates of P. pentosaceus found in the fermentation process of atole agrio have proven to be effective in the food industry, mainly as starter cultures in the fermentation of amaranth sourdoughs in addition to being an important microorganism in mycotoxin reduction during the fermentation of maize to produce Ogi (cereal porridge from Nigeria). The presence of this LAB was reported in the fermentation of atole agrio made from “de dobla” maize in the state of Tabasco, Mexico [51], along with other LAB such as Lp. plantarum, Lc. lactis, and Leuconostoc pseudomensenteroides. Hernandez-Vega [52] used these bacteria, in addition to P. pentosaceus, as starter cultures in the elaboration of white maize atole agrio. The P. pentosaceus isolate increased lactic acid concentration in all tests, while Lc. lactis showed lower lactic acid levels compared to the others. They also showed that LAB counts were higher at the end of the fermentations, significantly reducing the presence of enterobacteria, coinciding with the results obtained in this work where the reduction in the levels of these bacteria was achieved at the end of the fermentation process. This may confirm the presence of secondary metabolites such as bacteriocins (pediocins), which are small peptides (36 to 48 amino acid residues), capable of efficiently inhibiting bacteria such as L. monocytogenes [53,54].

The beneficial features of the genus Pediococcus, including its role as probiotic, has been previously reviewed [55], and the presence of P. pentosaceus in other fermentations involving cereals such as wheat and sorghum has been reported before [23,24]. The effect of starter cultures prepared from P. pentosaceus and Lc. lactis on the sensory properties and microbiological quality of white maize atole agrio has also been studied [56]. Recent investigations have also documented additional probiotic properties of various strains of P. pentosaceus, emphasizing its antioxidant properties, gut health enhancement, immune system modulation, cholesterol-lowering effects, and improved nutrient bioavailability. The studies on probiotic potential work as integrative approaches supporting the use of P. pentosaceus as a probiotic starter culture in food fermentation for the future development of precision fermentation processes and next-generation functional foods. [57].

Additionally, blue maize has been shown to have important phytochemical content such as ferulic and coumaric acids; anthocyanidins (cyanidin and pelargonidin) with antioxidant properties such as free radical inhibitors that protect cells against oxidative damage; in vitro anti-proliferative activity against cancer cells; and a protective effect on cardiovascular disease development and against risk of diabetes and cognitive function disorders. Additionally, blue maize phenolic compounds have been associated with modified starch digestion, showing a good positive correlation with slowly digestible and resistant starch fraction contents [2]. The presence of these phenolic compounds and the presence of potentially probiotic LAB are the basis for considering blue maize atole agrio as a functional food.

4. Conclusions

This study reports the isolation, analysis of probiotic potential, and identification of LAB from the fermentation of blue maize atole agrio from Tlapacoyan, Veracruz, Mexico. Pediococcus pentosaceus, predominant in the fermentation process of atole agrio, exhibited remarkable resistance to fluctuations in temperature (22–37 °C), pH, salt concentration, and bile salts. Additionally, adhesive characteristics such as coaggregation, autoaggregation, and cell wall hydrophobicity, as well as antibacterial and amylolytic activities were observed, supporting their probiotic potential. Consequently, a functional beverage loaded with potentially probiotic LAB and antioxidant phenolic compounds (already known to be present in blue maize) is produced during the blue maize atole agrio fermentation process, offering health-promoting benefits.