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Article

Ethnobiotechnological Analysis of the Lactic Bacterial Diversity in the Mezcal Fermentation of Four Palenques in Oaxaca, Mexico

by
Claudia López-Sánchez
1,
Víctor Adrián Espinoza-Martínez
1,
Felipe de Jesús Palma-Cruz
1,
Raúl Enríquez-Valencia
1,
Marcos Pedro Ramírez-López
1,
Lourdes Yaret Ortiz-Cortés
2 and
Peggy Elizabeth Álvarez-Gutiérrez
3,*
1
Tecnológico Nacional de Mexico, Instituto Tecnológico de Oaxaca, Avenida Ing. Víctor Bravo Ahuja No. 125 Esquina Calzada Tecnológico, Oaxaca de Juarez 68030, Oaxaca, Mexico
2
Escuela de Ciencias Químicas, Benemérita Universidad Autónoma de Chiapas, Carr. Panamericana Ocozocuautla-Cintalapa Km. 2.5., Ocozocuautla de Espinosa 29140, Chiapas, Mexico
3
SECIHTI, Tecnológico Nacional de Mexico, Instituto Tecnológico de Tuxtla Gutiérrez, Carretera Panamericana km 1080, Col. Juan Crispín, Tuxtla Gutiérrez 29050, Chiapas, Mexico
*
Author to whom correspondence should be addressed.
Fermentation 2026, 12(2), 89; https://doi.org/10.3390/fermentation12020089
Submission received: 18 November 2025 / Revised: 20 January 2026 / Accepted: 23 January 2026 / Published: 4 February 2026
(This article belongs to the Special Issue Women’s Special Issue Series: Fermentation)
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Abstract

Mezcal is an ancestral beverage from Oaxaca, Mexico, produced using traditional biotechnological procedures without artificial inoculation. The role of bacteria in fermentation has been studied less. Our work aimed to analyze the bacterial diversity in mezcal must and determine its relationship with ethnobiotechnological variables. Fermentation must samples were collected from four Palenques in the Valles Centrales, Cañada, and Mixteca regions of Oaxaca. Molecular and phenotypic identification and characterization, including ethanol tolerance testing, were performed. Ethnobiotechnological data were acquired through interviews and official databases. Six bacterial genera were identified in mezcal must, including Lactiplantibacillus, Enterobacter, Enterococcus, Bacillus, Leuconostoc, and Levilactobacillus. Therefore, through a factorial experiment, it was determined that the selected Lactiplantibacillus plantarum strains were able to withstand 6% and 8% ethanol. A direct relationship was observed between species diversity and the degree of population marginalization. Palenque 2, representing the most urbanized population, exhibited the lowest diversity, with only two species detected. In contrast, Palenques characterized by high marginalization harbored a substantially greater diversity, ranging from six to ten species. These differences are likely associated with variations in microbiota composition, microbial growth dynamics, and ethanol tolerance. Overall, the findings suggest that ethnobiotechnological factors play a significant role in shaping bacterial diversity. In Oaxaca, “ethnobiotechnological” implies the deep relationship between biodiversity, Indigenous culture, and territory, with a strong emphasis on living traditional knowledge and its intergenerational transmission.

1. Introduction

Fermented beverages, such as mezcal, are still produced using traditional or ancestral methods. Compared to pulque, tequila, and bacanora, mezcal features the most diverse and functionally complex ecosystem of lactic acid bacteria, which explains its wide sensory variability and close relationship with geographic origin and traditional practices [1,2].
This biotechnological process lacks artificial inoculation; therefore, the microbiota responsible for agave fermentation is not a single strain. The microbiota responsible for ethanol production and the organoleptic characteristics of mezcal is a spontaneous combination of bacteria and yeasts. These microorganisms have developed complex metabolisms and intricate ecological interactions [3]. This ancestral drink is culturally connected to the native Oaxacan people and has economic importance [4].
The production of mezcal is a complex biotechnological process described in [5]. A key unit operation in mezcal production is the fermentation of modified agave stems, which are inoculated with spontaneous microorganisms that produce a wide range of compounds, among them ethanol. The qualitative and quantitative composition of the must microbiota depends on the geographical and ecological region of the raw material, the biotechnological process, and physicochemical variables such as pH and temperature [6].
Bacteria linked to agave play an important role in fermentation, creating flavor and aroma compounds for mezcal [7]. Compared to yeasts, bacteria are less studied because isolating them from alcoholic fermentations is more time-consuming, which can limit species identification and further research.
Lactic acid bacteria (LAB) constitute a group of Gram-positive bacteria united by a constellation of morphological, metabolic, and physiological characteristics. In addition to being Gram-positive, LAB are non-sporing, non-respiring cocci or rods that produce lactic acid as the major product of carbohydrate fermentation. The LAB term is associated with bacteria involved in food and feed fermentation, including those commonly associated with the (healthy) mucosal surfaces of humans and animals. The boundaries of the group have been subject to some controversy, but historically, the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus form the core of this group [5].
Several studies have identified Lactiplantibacillus plantarum as one of the most frequent LAB during the fermentation of mezcal and other fermented agave products. Its presence is highly adaptable to environments with low pH, elevated ethanol concentrations, and limited nutrient availability [6]. Understanding its function is essential for explaining the process’s stability and the sensory complexity of the final product.
Enterobacter is known for improving soil and boosting crop growth [8,9]. As a well-studied plant growth-promoting rhizobacterium (PGPR), it enhances soil nutrients, regulates enzymatic activity, forms biofilms, increases resilience, and supports plant health under stress [10,11,12,13,14]. Additionally, Enterobacter has been found in agave tissues; for instance, E. hormaechei appears in roots and leaves of Agave tequilana and A. victoriae-reginae, suggesting its presence in raw material but not necessarily dominance during mezcal fermentation after cooking [15].
The genus Enterococcus is regularly found among lactic acid bacteria during mezcal fermentation, as identified through both microbiological and molecular methods. Enterococcus is a Gram-positive, catalase-negative, and fermenting bacteria that can produce lactic acid from carbohydrates, which contributes to the acidification of agave must during the fermentation’s early stages [1,5,14]. Enterococcus’s participation in mezcal fermentation mainly involves pH reduction, creating a less favorable environment for contaminating microorganisms and promoting the growth of fermentative yeasts, such as Saccharomyces cerevisiae and non-Saccharomyces yeasts [1,16]. This reduction in pH can improve process stability and reduce the proliferation of undesirable bacteria.
Additionally, Enterococcus species exhibit notable enzymatic activities, including proteases and glycosidases, which may be secreted.
Aroma precursors from cooked agave, along with secondary metabolites such as organic acids and volatile compounds, help shape the sensory profile of mezcal [5].
Culture-independent analysis removes the biases present in culture-dependent methods, improving accuracy in identifying microbial communities [17]. In San Luis Potosí, lactic acid bacteria (LAB) were predominant, while studies in Oaxaca detected LAB, acetic acid bacteria (AAB), and other bacteria involved in mezcal fermentation [6,18]. Evidence suggests regional variations in bacterial populations, such as LAB and AAB, influence ethanol and organic acid production, affecting Mezcal’s unique flavor profile; at high concentrations, some metabolites are undesirable but may act as precursors to desirable compounds like esters [1,17]. Lactobacillus plantarum adapts well to various environments by fermenting diverse carbohydrates, and several bacteria from traditional drinks show probiotic potential. Fermented beverages often contain Lactococcus, Lactobacillus, Leuconostoc, Oenococcus, and Streptococcus; Lacticaseibacillus casei and paracasei are common both in nature and the gut microbiota [6,15]. Native fermented beverages are globally recognized heritage items, yet Oaxacan mezcal faces a decline in biotechnological knowledge and tradition preservation. This study aimed to analyze bacterial diversity in mezcal must and its relationship with factors like climate, land use, vegetation, Indigenous population, and socioeconomic conditions. Biodiversity loss near Palenques has decreased microbial populations, reducing the complexity of aromas and flavors in artisanal mezcal.

2. Materials and Methods

2.1. Sampling, Isolation, and Propagation of Bacteria

Selecting the four Palenques was based on a comparative sampling strategy to capture the ethnobiotechnological, environmental, and sociodemographic heterogeneity of Oaxacan mezcal production. The Palenques are in three distinct mezcal-producing regions (Valles Centrales, Cañada, and Mixteca), which differ in altitude, hydrographic basins, land use, vegetation cover, and climatic conditions. Additionally, their municipalities possess socio-economic contexts ranging from extremely low to high marginalization index and differences in urbanization levels and agricultural activity.
In this study, Palenques are not merely production sites but are ethnobiotechnological units where spontaneous fermentation occurs. They integrate environmental conditions, traditional practices, raw materials, and microbial communities. Therefore, Palenques constitute the fundamental comparative framework for understanding how bacterial diversity in mezcal fermentation is shaped by regional and socio-environmental factors.
The design allowed evaluation of how regional and ethnobiotechnological variables influence bacterial diversity during spontaneous mezcal fermentation. Rather than statistical representativeness, the study focuses on biological and cultural representativeness, reflecting the diversity of Oaxacan traditional mezcal production systems. Similar purposive approaches have been employed in previous studies regarding microbial diversity in traditional fermentations, where ecological and cultural variability are central to understanding microbiota dynamics.
Agave angustifolia is a widespread and adaptable species, both cultivated and wild. It is primarily grown in Oaxaca for mezcal, especially in Tlacolula, Etla, and Nochixtlán for distilleries 1, 2, and 4. The species is intercropped with staple crops or other agaves, tolerates poor conditions, and grows on steep former ecosystem slopes near distilleries 1 and 4. At distillery 2, it has been extensively cultivated for over three generations.
The stems of mature plants used in artisanal mezcal production typically weigh between 60 and 80 kg. On the other hand, Agave potatorum is a wild agave species that reproduces exclusively by seed and has been used in mezcal production since the late twentieth century. It grows naturally among xerophytic scrubland and oak forests, where it is harvested. It is commonly known as tobalá or papalometl. In addition to being a raw material for mezcal, its flowers are eaten and its leaves are used in traditional medicine. Its high content of total reducing sugars has made it one of the preferred agaves for artisanal mezcal production in Oaxaca.
The fermentation must samples were aseptically collected directly from the fermentation container in each of the four Palenques. The pH was measured with a Hanna Instruments HI221 instrument, which had been previously calibrated. Total suspended solids expressed in Brix degrees were measured with a digital monocular ABBE refractometer model ZWAJ.
The study sites were as follows: Palenque 1, located in the municipality of San Dionisio Ocotepec at 1620 meters above sea level(m.a.s.l) (16°46′11″ N, 96°22′11″ W); Palenque 2 in San Pablo Etla at 1726 m.a.s.l. (17°09′14″ N and 96°44′49″ W); and Palenque 3 in Concepción Buenavista at 1734 m.a.s.l. (17°57′43″ N and 97°27′25″ W). Finally, Palenque 4 corresponded to the municipality of San Pedro Teozacoalco at 1586 m.a.s.l. (17°01′01″ N and 97°17′12″ W). The temperature, pH, and total soluble solids of each fermentation container were determined in situ. The samples were refrigerated at 4 °C until processing. Most samples were sent to the laboratories of the National Technological Institute of Mexico on the Oaxaca and Tuxtla Gutiérrez campuses. The sampling sites of the four Palenques were the same as reported in [19].
Each 1 mL of must juice sample was homogenized with 0.1% of peptone solution. The 1 × 10−6 dilutions of each sample were inoculated in MRS medium (supplemented with nistatine 0.002%) by a spread plate in Petri dishes and incubated at 30 °C for 120 h. Single isolated colonies were reseeded until pure cultures of bacterial strains were obtained.
Selective media were used for phenotypic genera determination, such as Lactobacillus selection (LBS) for Lactobacillus spp. [20,21], M17 media for Lactococcus spp. [22], and MSE media for Leuconostoc spp. [23,24]. The Elliker media was placed in Petri dishes, and lactic acid bacteria were inoculated to be incubated at 33 to 35 °C for 18 to 48 h. Significant bacterial growth indicated the presence of Lactobacillus and Streptococcus genera. The LAB strains were inoculated in the M5 medium and incubated at 30 °C for 48 h. They were considered homolactic LAB if the blue medium turned yellow and heterotactic LAB if the medium remained blue [25].

2.2. DNA Extraction, PCR Amplification, and Phylogenetic Analysis

Genomic DNA was extracted from frozen cell stocks using the ZR Fungal/Bacterial DNA kit (Zymo Research, Irvine, CA, USA; catalog #D6005) following the manufacturer’s instructions. The 16S rRNA gene was amplified through the polymerase chain reaction (PCR) technique with the oligos 8F (5′–AGAGTT TGATCCTGGCTCAG–3′) and 1492R (5′–GGTTACCTT GTTACGACTT–3′). The PCR conditions were performed as described in [26]. The sequence analysis used the same primers (Macrogen, Seoul, Republic of Korea, and the sequences were edited in the BioEdit software package version 7.2.5. These sequences were compared in the GenBank database at the National Center for Biotechnology Information (NCBI) using the nucleotide Blast tool (version 2.17.0, updated 22 July 2025) [27]. The consensus sequences were aligned using the on-line tool CLUSTALW version 2.0. The alignment helped construct a phylogenetic tree under the maximum parsimony model, with a bootstrap analysis of 1000 replications [28]. The MEGA 11 software package version 11.0.13 performed the analyses [29].

2.3. Phenotypic Characterization of Bacterial Isolates

Morphological and biochemical tests were conducted for all strains. Cell morphology and Gram staining, as described by [30], were used for macroscopic characterization; purified colonies were observed under a stereomicroscope to assess the shape, size, color, elevation, and other features of each selected strain. Microscopic characterization was performed using a smear and Gram stain. The phenotypic assays for all isolates included catalase and aerobic/anaerobic metabolism [31] as fermentation type in M5 medium [32]. The type of fermentation (homofermentative or heterofermentative) was found according to [33].
Fifteen differential techniques were used in the biochemical tests to evaluate carbohydrate metabolism, as outlined in [34]. The nitrate reduction capacity was evaluated with nitrate broth, as defined by [35,36]. The gas formation test in MRS and the catalase assay were conducted as described in [37].

2.4. Tolerance Resistance Experiment

From an applied perspective, ethanol tolerance is a critical factor in optimizing fermentation processes in the food, pharmaceutical, and energy industries. Identifying and characterizing organisms or strains with greater tolerance can improve the productivity, stability, and efficiency of these processes, particularly in the production of bioethanol, where the accumulation of the final product limits the performance of the biological system [38].

Strategy for Studying Tolerance to Ethanol

The experimental strategy was based on a controlled, comparative design that allowed evaluating the effect of ethanol as a chemical stress agent on lactic acid bacteria isolated from mezcal fermentation. These bacteria were part of the microbiota associated with traditional mezcal fermentative processes and were naturally exposed to progressive increases in ethanol, making them a relevant biological model for studying mechanisms of tolerance to alcoholic stress [39].
The MRS broth as a culture medium was justified by its optimal nutritional composition for the growth of lactic acid bacteria, thereby minimizing nutritional limitations and ensuring reproducible conditions throughout the experimental development. In this way, the observed effects could be primarily attributed to ethanol [40].
In the experimental design, ethanol concentration was established as the independent variable, whereas bacterial growth and viability were considered dependent variables. Including a gradient of ethanol concentrations and an unexposed control let us evaluate a dose-dependent response and identify tolerance thresholds characteristic of lactic acid bacteria associated with mezcal fermentation.
The concentrations of 6% and 8% ethanol were selected because they represent levels of alcoholic stress during distinct stages of mezcal fermentation. 6% corresponded to sublethal stress, whereas 8% represented severe stress, which allowed [38].

2.5. Ethnobiotechnological Data Acquisition and Analysis

Data on mezcal production processes were obtained from mezcaleros at four Palenques through personal, unstructured interviews for people’s oral consent. The Palenques are the traditional factories for their production, and sociodemographic data was obtained from official databases [39]. To understand the biodiversity of the LAB, social, economic, cultural, and environmental indicators were used to recognize their influence on the bacterial presence in each Palenque [41,42]. To confirm what was observed, the Shannon-Weiner index [43], the Chao index (1984) [44], and the Pearson correlation coefficient (1895) [45] were applied, in addition to calculating the relative frequency and abundance of the isolated and identified strains in each Palenque.

3. Results

3.1. Molecular and Phenotypic Identification of Bacteria in the Must of Mezcal Fermentation

Strains were identified with the 16S rRNA gene marker (Table 1) or biochemical markers (Table 2 and Table S1). The biochemical determination was analyzed with specialized literature. The Lactiplantibacillus and Lacticaseibacillus species were based on [46]. The positive acid clot reaction in Litmus milk Microbiolo48 indicated Lactiplantibacillus pentosus (P1OAXL1, P1OAXL2, P1OAXL4, P1OAXL9), whereas an adverse response in Litmus milk medium was indicative of Lactiplantibacillus plantarum. The Lactiplantibacillus genus was P1OAXL5, P1OAXL7, P1OAXL8, P3OAXL19, P3OAXL20, P4OAXL22, and P4OAXL26 strains were assigned based on BLAST results, and the species were identified as L. plantarum through biochemical tests. Negative mannitol fermentation was indicative of Enterobacter sichuanensis (P4OAXL61) according to [47,48]. P4OAXL11 and P4OAXL27 (Bacillus sp.), P4OAXL62, P4OAXL66 (Enterobacter sp.), P3OAXL15 (Lactiplantibacillus spp.), and P4OAXL64 (Lacticaseibacillus) were determined only for genera.
Six genera were identified in most mezcal fermentations: Lactiplantibacillus, Enterobacter, Enterococcus, Bacillus, Leuconostoc, and Levilactibacillus (Table 1). Two species of Bacillus (amyloliquefaciens and pumilus), one species of Enterobacter sichuanensis, two species of Enterococcus (faecalis and gallinarum), three species of Lactiplantibacillus (plantarum, pentosus, and diolivorans), one species of Leuconostoc mesenteroides, and one species of Levilactobacillus brevis were determined.

3.2. Phylogenetic Analysis of Isolates of Must

The phylogenetic analysis of isolates is represented in Figure 1. The maximum parsimony phylogenetic tree shows that the species identified from Palenques 1, 2, 3, and 4 form a cluster grouped by genus. In the first clade is the genus Lactiplantibacillus, including the species plantarum, pentosus, and diolivorans. The genus Bacillus and its species, such as amyloliquefaciens and pumilus, represent the second clade. The P4OAXL24 strain of Leuconostoc mesenteroides is grouped in a single branch but shares the same cluster as the Enterobacter genus. The genus Enterococcus determines the next cluster and has two species: E. faecalis and E. gallinarum. Finally, the only species separated from all the samples is the P3OAXL16 strain, which belongs to Staphylococcus sp. probably because it is a Gram-positive bacterium. As shown in the figure, although diversity is low, its phylogenetic signal is clear. The species related to Palenque 2 are differentiated by their phylogenetic signal and their reduced representation.
We employed the selective media MRS, M17 and M5 to isolate and differentiate lactic acid bacteria according to their physiological and metabolic requirements. MRS is the most widely used medium for the general isolation of lactobacilli because of its acidity and accompanying microbiota-inhibitory agents. Furthermore, M17 is vital in studying Lactococcus and Streptococcus, especially in the dairy industry, as it better simulates the growing conditions of starter cultures. M5, on the other hand, promotes heterofermentative lactic acid bacteria involved in aroma and gas production. The combined use of these media improves the microbiological characterization of fermented foods and dairy products [49].
Selective media defines which bacteria grow, whereas 16S rRNA analysis defines who they really are. Together, both approaches strengthen the identification, ecological study, and technological application of lactic acid bacteria in fermented foods and dairy products [50].

3.3. Ethanol Tolerance

The growth rate depends on the strain, but the isolates from Palenque 1 grew faster than the others: P1OAXL1 (Lactiplantibacillus pentosus) and P1OAXL10 (Bacillus pumilus). Differences in growth rates were also evident among strains originating from different Palenques, suggesting that local fermentation conditions and microbial adaptation may influence bacterial growth dynamics. The observed strain-level variability highlights the importance of considering individual isolates rather than species-level classifications when evaluating microbial performance in mezcal fermentations. The highest microbial growth in MRS media was selected and grown under alcohol fermentation conditions (Figure 2, Table 3). The selected strains were subjected to a rapid ethanol tolerance test in MRS medium at 0%, 6%, and 8% ethanol concentrations in test tubes. Four strains were selected for growth kinetics: P1OAXL2 Lactiplantibacillus pentosus, P1OAXL5 Lactiplantibacillus plantarum, P3OAXL19 Lactiplantibacillus plantarum, and P3OAXL20 Lactiplantibacillus plantarum. All four strains grew in 6% and 8% ethanol in MRS medium, and growth was 2 to 4 times lower than in the absence of ethanol. P3OAXL19 Lactiplantibacillus plantarum had the highest growth rate in ethanol. None of them grew by 10% ethanol.

3.4. Bacterial Diversity and Ethnobiotechnological Context

Throughout human history, biodiversity has varied in response to human activities, and vice versa. Multiple research efforts, spanning from Darwin’s era to the present, consistently substantiate this coexistence. However, it is still uncertain whether variations in the microbial diversity of fermented beverages are influenced by sociodemographic or ethnobiotechnological factors (Table 4).
The weather in the region of the four Palenques is similar, and the average temperature differs by 4 degrees between them. The average rainfall is also identical. Palenques 2 in San Pablo Etla and Palenque 4 in San Pedro Teozacoalco) belong to the Atoyac River basin, although Palenque 2 is in the Central Valleys, and Palenque 4 is in the Mixteca region. In contrast, there are significant differences in the agricultural activity of the municipalities where the Palenques are located since Palenque 2 and 4 have significantly higher levels of farming activity (23.03% and 23.10%, respectively). Moreover, Palenque 1 is close to 10%, and Palenque 3 is less than 1%.
The four municipalities of the Palenques have an urban population of less than 17%. Furthermore, Palenques 1, 3, and 4 have an urban population of less than 2%. Therefore, the four study sites are in rural areas. It is essential to highlight the significant difference between Palenque 2 and the others, given the degree of marginalization. Palenques 1, 3, and 4 have a high degree of marginalization, and poverty levels range from 73.93% to 99%, indicating that the entire population lives in poverty and marginalization. The diversity of species found in each Palenque may be related to the degree of population marginalization.
According to the values obtained from applying the Chao coefficient, there is an inversely proportional relationship between the richness of LAB and Palenques 3 and 4 (7.25 and 5.0, respectively), (Table 4). Note that both Palenques are in regions with prominent traditional practices in natural resource management and in low-intensity agriculture with less agrochemical use. Therefore, they preserve a richer, more diverse microbiota. This coefficient also helped identify that moderate water stress combined with altitude, especially in Palenque 3 (at 1600 m.a.s.l and semiarid), could promote the coexistence of multiple resistant strains, rather than a dominant single LAB strain.
After calculating the Shannon-Weiner index, it was determined that Palenque 4, from which eight LAB strains were isolated, exhibits the highest richness (H′ = 2.16). It is the site with the highest biological diversity in the study, with five unique species: Enterobacter sichuanensis, Enterococcus gallinarum, Lactiplantibacillus diolivorans, Leuconostoc mesenteroides, and Levilactobacillus brevis. Thus, it is a Palenque with a complex, non-standardized fermentative ecosystem: ecologically, it has the most stable and diverse microbiome, making it potentially unique for bioprospecting unconventional strains. Palenques 3 and 1, on the other hand, with low diversity (H′ = 1.05 and 0.94, respectively), harbor microbiomes dominated by LAB from the genera Bacillus and Lactiplantibacillus. Finally, the lowest diversity was observed in Palenque 2 (H′ = 0.69), possibly because of minimal ecological complexity under anthropogenic pressure because it has the most urbanization.
After analyzing the correlation between the different indicators shown in Table 4 and applying the Pearson coefficient (r), a positive correlation (r = 0.94) was found between the presence of Bacillus amyloliquefaciens and the region with the highest percentage of intensive agriculture, such as that practiced around Palenque 2. It was also confirmed that rainfall favors the diversity of LAB (r = 0.91) and directly determines microbial richness, notably visible in Palenque 4. Furthermore, Palenques 1 and 4, which are in the municipalities with the highest poverty index, maintain larger populations of LAB (r = 0.75).
The most frequently isolated species, based on their presence in three of the four Palenques (Table S2), were Lactiplantibacillus plantarum, followed by Bacillus amyloliquefaciens. Regarding taxonomic diversity, Palenque 4 is represented by six genera and seven species among the 11 LAB found in this study. The most notable aspect is that five of these species appeared only in this Palenque, and the remaining two correspond to the most frequent LAB. From Palenque 3, five strains were isolated, belonging to three different genera and three different species; from Palenque 1, ten strains were isolated, belonging to two genera, three species, and one subspecies; and finally, from Palenque 2, two strains were obtained, belonging to two different genera and two different species (Table S2).

4. Discussion

Mezcal is an iconic beverage from the Mexican state of Oaxaca. It comes from fermenting various agave species. The process involves five stages: harvesting the agaves, cooking, crushing, fermentation, and distillation. The fermentation process is critical because it requires yeast and bacteria. Initially, yeast transforms the agave sugars into ethanol and CO2, producing secondary metabolites such as esters and aldehydes. These are responsible for mezcal’s distinctive flavor [50].
Previously, we identified the yeast community involved in fermenting mezcal at four Oaxacan Palenques. Now, we focus on the bacteria that ferment most mezcal. They belonged to the genera Bacillus, Enterobacter, Enterococcus, Lactiplantibacillus, Lacticaseibacillus, Leuconostoc, Levilactobacillus, and Staphylococcus (Table 1, Table 2 and Table S1).
Although the fermentation of beverages such as mezcal and tequila ends with a distillation stage that removes most live microorganisms, the study of LAB in the fermentation phase is still relevant to aromatic profiles that are transferred through precursor metabolites before distillation, the consistency and reproducibility of fermentation, and the control of faulty processes (e.g., pH deviations or unwanted metabolites). In comparison, non-distilled beverages such as pulque retain active microbiota, so the LAB study there also provides information on potential functional and structural benefits of microbiota. These contrasts allow us to discuss how LAB influences distinct types of agave beverages (fermented versus distilled) [51].
During mezcal fermentation, bacteria from the Lactobacillus group, currently reclassified into genera such as Lactiplantibacillus, Lacticaseibacillus, and Limosilactobacillus, help biochemically transform the agave must. These microorganisms metabolize fermentable sugars and produce lactic acid, which progressively lowers the pH. This acidification enhances the microbiological stability of the fermentation system, limits the growth of undesirable microorganisms, and influences the sensory perception of the final product [5].
In addition to lactic acid, some heterofermentative species generate acetic acid and, to a lesser extent, other organic acids such as succinic and formic that contribute to the chemical complexity of the must and can provide taste sensations. Furthermore, although Lactobacillus is not primarily responsible for alcohol production, it can synthesize insignificant amounts of ethanol and other secondary metabolites that interact with yeast-produced compounds during the mixed fermentation characteristic of mezcal.
A relevant aspect of the metabolic activity of these bacteria is the indirect formation of esters, especially ethyl lactate, resulting from the reaction between lactic acid and ethanol. This compound is essential from a sensory standpoint, as it provides smooth, creamy, and slightly fruity notes that are typically associated with artisanal mezcals. Similarly, Lactobacillus can produce carbonyl compounds such as diacetyl and acetaldehyde, which, depending on their concentration, provide buttery or sweet aromas.
The Bacillus genus present in the residual mezcal must from production is frequently observed. Its function is to produce enzymes that hydrolyze the polysaccharides and proteins in agave. Consequently, monosaccharides are produced and fermented by yeast. Bacillus can also perform nitrogen fixation and amino acid biosynthesis, generating assimilable nitrogen that enables yeast to perform fermentation. Another reason for Bacillus in mezcal production is its ability to grow under acidophilic and thermophilic conditions, as well as in the presence of ethanol [52].
The presence of Enterobacter should be interpreted as part of the transient microbial community characteristic of spontaneous mezcal fermentation, where nondominant bacteria can ecologically and metabolically influence the roles during early fermentation stages before being eliminated by increasing ethanol concentrations and acidic conditions.
Moreover, the presence of Enterobacter bacteria can be explained by their ability to secrete fermentative enzymes, such as pyruvate formate lyase and lactate dehydrogenase, which convert sugars into organic acids, ethanol, and gases [53]. In addition to secreting enzymes such as urease, they release ammonia and amino acids into the must, generating assimilable nitrogen that will later be utilized by yeast and lactic acid bacteria. They also employ pathways for synthesizing acetoin, 2,2-butanediol, succinate, and short-chain fatty acids, which contribute to mezcal’s aromatic profile. Furthermore, Figure 1 shows the phylogenetic relationships among the bacteria isolated from the four Palenques. Specifically, the bacteria from Palenque 3 consistently form clades with the bacteria isolated from Palenques 1, 2, and 4. This may be caused by the biological characteristics shared by these sites, particularly water, soil, and air, as well as the biochemical and metabolic functions performed synergistically by these microorganisms.
For example, the distinct species of Lactiplantibacillus encode genes associated with carbohydrate metabolism, stress tolerance, and exopolysaccharide production [54]. They contribute to the acidification of must and produce antimicrobial compounds. Bacteria of the genus Bacillus can adapt to different environments [6,55], perhaps because of their ability to form spores. Their key function is the secretion of hydrolases that degrade fermentable carbohydrates and amino acids. The presence of Enterococcus may signal contamination. Conversely, it also contributes to mezcal’s flavor.
There are no published studies so far that indicate Staphylococcus plays a natural or beneficial role in the fermentation of mezcal. Conversely, microbiota studies report lactic acid bacteria and fermenting yeasts. In traditional alcoholic [16], in the fermentation of other beverages such as beer, Staphylococcus has been isolated as a contaminant in some cases but not as a primary fermenter [55].
Former studies of yeast diversity in Palenques in Oaxaca demonstrated that yeasts are responsible for alcoholic fermentation [19]. Furthermore, it was shown that differences in the biotechnological process influence fungal diversity. In this study, the differences between the four Palenques were related to ethnobiotechnological variables outside the Palenque walls. It was suggested that differences in the degree of marginalization and socio-environmental characteristics influence bacterial diversity during the mezcal fermentation process. The diversity of species found in each Palenque is related to the degree of urbanization of the population. Palenque 2, which has the most significant urban population (16.87%), has the lowest diversity of bacterial species [2]. In contrast, in Palenques where the urban population is less than 2%, the number of bacterial species triples or quintuples.

5. Conclusions

The raw materials, microbiota, and human intervention have enabled the fermentation of alcoholic beverages like mezcal. In Oaxaca, the world’s birthplace of mezcal, Palenques are the traditional factories for its production. This fermentation process still maintains a syncretism between biotechnology and the worldview of mezcal producers. Therefore, at least for the expert mezcaleros of Oaxaca, there is an intimate connection between the production process and cultural rituals, resulting in a fermented beverage with a unique flavor. Each mezcal produced in the Palenques shows perceptible differences even to the most discerning palates. In this study, we demonstrate that mezcal fermentation in four distilleries in Oaxaca exhibits differences in microbiota. While the species found are similar in some distilleries, there are differences in growth and ethanol tolerance.
Furthermore, we also demonstrate that the regional and biotechnological processes may be associated with the diversity of bacteria involved in fermentation. The microbiota that produces mezcal depends on both yeasts and bacteria. The latter is crucial in the fermentation process and, consequently, in the flavor.
Compared to pulque, tequila, and bacanora, mezcal features the most diverse and functionally complex ecosystem of lactic acid bacteria, which explains its wide sensory variability and close relationship with geographic origin and traditional practices. The study of LAB in mezcal is not only key for understanding its fermentation but also for designing conservation strategies for artisanal processes.

Supplementary Materials

The following supporting information can be downloaded at https://www.mdpi.com/article/10.3390/fermentation12020089/s1, Table S1: Phenotypic characterization of isolates from the must of mezcal in four Palenques in Oaxaca, Mexico; Table S2: Frequency and distribution of lactic acid bacteria strains isolated from four Palenque fermentation samples from Oaxaca, Mexico.

Author Contributions

C.L.-S. and F.d.J.P.-C. conceived, carried out personal interview with mezcaleros and provided the resources. C.L.-S., V.A.E.-M., F.d.J.P.-C. and P.E.Á.-G. designed this work and wrote the first draft of the manuscript. C.L.-S., V.A.E.-M., F.d.J.P.-C., P.E.Á.-G., R.E.-V. and M.P.R.-L. edited the figures. C.L.-S., F.d.J.P.-C., P.E.Á.-G. and L.Y.O.-C. wrote, review and edited the final version. All authors commented on the figures and previous version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by National Technological Instituto of México, for Project “Bacteriocinas de bacterias lácticas procedentes de la fermentación del mezcal y su potencial en la salud” M00-PR-03-R01, Proyecto 22216. Convocatoria 2025 de Proyectos de Investigación Científica, Desarrollo Tecnológico e Innovación by Claudia López Sánchez.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

rRNA 16S sequences of bacterial isolates are available in the GenBank database under the accession numbers listed in Table 1.

Acknowledgments

The authors thank the mezcaleros of the four Palenques for the facilities for sampling and informal interviews. The authors thank the students involved in data acquisition (Antonio de Jesus Paz, Adriana Palacios Jauri, Dulce Maria Villanueva Castillo and Keila Mirel Vasquez Agudo). While preparing this manuscript, the authors used diverse AI tools for editing the manuscript. The authors have reviewed and edited the manuscript and take full responsibility for its content.

Conflicts of Interest

The authors declare no conflicts of interest.

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Fermentation 12 00089 g001
Figure 1. Maximum parsimony phylogenetic tree of nucleotide sequences of bacterial strains isolated from four artisanal Palenques in Oaxaca, Mexico. Each branch corresponds to a resulting sequence obtained from one of the Palenques. The strain names are based on morphological and biochemical identification. The circles’ colors are as follows: Palenque 1 in San Dionisio, Ocotepec (green); Palenque 2 in San Pablo, Etla (red); Palenque 3 in Concepción Buenavista (blue); and Palenque 4 in San Pedro, Teozacoalco (purple). The phylogenetic tree was generated using an ultrafast bootstrap with 1000 replicates in MEGA version 11, with a probability for each region. L: Lactiplantibacillus, Lv: Levilactobacillus, B: Bacillus, Lu: Leuconostoc, E: Enterobacter, En: Enterococcus.
Figure 1. Maximum parsimony phylogenetic tree of nucleotide sequences of bacterial strains isolated from four artisanal Palenques in Oaxaca, Mexico. Each branch corresponds to a resulting sequence obtained from one of the Palenques. The strain names are based on morphological and biochemical identification. The circles’ colors are as follows: Palenque 1 in San Dionisio, Ocotepec (green); Palenque 2 in San Pablo, Etla (red); Palenque 3 in Concepción Buenavista (blue); and Palenque 4 in San Pedro, Teozacoalco (purple). The phylogenetic tree was generated using an ultrafast bootstrap with 1000 replicates in MEGA version 11, with a probability for each region. L: Lactiplantibacillus, Lv: Levilactobacillus, B: Bacillus, Lu: Leuconostoc, E: Enterobacter, En: Enterococcus.
Fermentation 12 00089 g001
Fermentation 12 00089 g002
Figure 2. Growth rates (h−1) of lactic acid bacteria isolated from four mezcal Palenques in Oaxaca, Mexico. Each point represents an individual strain. The color of the bubbles corresponds to Palenque 1 in San Dionisio, Ocotepec (green), Palenque 2 in San Pablo, Etla (red), and Palenque 3 in Concepción Buenavista (blue), Palenque 4 in San Pedro, Teozacoalco (purple). Bubble size reflects relative growth performance.
Figure 2. Growth rates (h−1) of lactic acid bacteria isolated from four mezcal Palenques in Oaxaca, Mexico. Each point represents an individual strain. The color of the bubbles corresponds to Palenque 1 in San Dionisio, Ocotepec (green), Palenque 2 in San Pablo, Etla (red), and Palenque 3 in Concepción Buenavista (blue), Palenque 4 in San Pedro, Teozacoalco (purple). Bubble size reflects relative growth performance.
Fermentation 12 00089 g002
Table 1. Identification of bacterial strains isolated from mezcal must in four Palenques in Oaxaca, Mexico, based on 16S rRNA gene sequencing and conventional biochemical tests.
Table 1. Identification of bacterial strains isolated from mezcal must in four Palenques in Oaxaca, Mexico, based on 16S rRNA gene sequencing and conventional biochemical tests.
StrainQuery Cover%
Ident		E-ValueGene Bank Accession Number
NBRC 15535 Bacillus amyloliquefaciens T MK182997.1
P2OAXL11 Bacillus amyloliquefaciens97%87.91%0.0PP694281
P3OAXL18 Bacillus amyloliquefaciens100%98.51%0.0PP694284
P3OAXL21 Bacillus amyloliquefaciens99%93.78%0.0PP694287
P4OAXL25 Bacillus amyloliquefaciens99%98.47%0.0PP694290
ATCC 7061 Bacillus pumilus T KY034389.1
P1OAXL10 Bacillus pumilus93%78.02%3.00 × 10−63
P4OAXL11 Bacillus sp. ****PP694281
P4OAXL27 Bacillus sp. ****
WCHECL1597 Enterobacter sichuanensis T MG832788.1
P4OAXL61 Enterobacter sichuanensis100%97.41%0.0PP694294
P4OAXL62 Enterobacter sp. ****PP694295
P4OAXL66 Enterobacter sp. ****PP694297
ATCC19433 Enterococcus faecalis T NR_115765.1
P2OAXL14 Enterococcus faecalis100%99.93%0.0PP694282
P3OAXL17 Enterococcus faecalis99%99.35%0.0PP694283
ATCC 49573 Enterococcus gallinarum T LC097066.1
P4OAXL29 Enterococcus gallinarum100%99.62%0.0PP694293
JCM 1149 Lactiplantibacillus pentosus T LC064896.1
P1OAXL1 Lactiplantibacillus pentosus99%99.42%0.0PP694274
P1OAXL2 Lactiplantibacillus pentosus100%90.65%0.0PP694275
P1OAXL4 Lactiplantibacillus pentosus99%99.78%0.0PP694276
P1OAXL9 Lactiplantibacillus pentosus100%99.86%0.0PP694280
CIP 103151 Lactiplantibacillus plantarum MN326667.1
P1OAXL3 Lactiplantibacillus plantarum90%91.67%6 × 10−53
P1OAXL5 Lactiplantibacillus plantarum ****
P1OAXL6 Lactiplantibacillus plantarum subargentoratensis100%99.50%0.0PP694278
P1OAXL7 Lactiplantibacillus plantarum ****PP694279
P1OAXL8 Lactiplantibacillus plantarum79%97.40%0.0PP694280
P3OAXL19 Lactiplantibacillus plantarum ****PP694285
P3OAXL20 Lactiplantibacillus plantarum ****PP694286
P4OAXL22 Lactiplantibacillus plantarum ****PP694288
P4OAXL26 Lactiplantibacillus plantarum ****PP694291
JCM 12183 Lactiplantibacillus diolivorans T LC311745.1
P4OAXL65 Lactiplantibacillus diolivorans98%97.75%0.0PP694296
P3OAXL15 Lactiplantibacillus sp. ****
P4OAXL64 Lacticaseibacillus sp. ****
ATCC 8293 Leuconostoc mesenteroides T NR_074957.1
P4OAXL24 Leuconostoc mesenteroides99%99.19%0.0PP694289
ATCC 14869 Levilactobacillus brevis T NR_116238.1
P4OAXL28 Levilactobacillus brevis99%96.48%0.0PP694292
Bacterial strains were isolated from mezcal must samples collected from four artisanal Palenques in Oaxaca, Mexico. Molecular identification was performed by partial sequencing of the 16S rRNA gene, followed by comparison with reference sequences available in public databases. Biochemical identification was conducted using conventional tests, including carbohydrate fermentation profiles, catalase activity, and other physiological and biochemical assays. Strain codes indicate the Palenque of origin and isolate number. * Species determination with the biochemical markers. T Type strain in bold.
Table 2. Biochemical characterization of bacterial isolates from mezcal must collected in four Palenques in Oaxaca, Mexico.
Table 2. Biochemical characterization of bacterial isolates from mezcal must collected in four Palenques in Oaxaca, Mexico.
GroupSpecieStrainGAL aGLU aFRU aMAN aLAC aSUC aGAS bNO3 cLM dLBS eM17 fELLI fMSE fCAT gGRAM h
1Lactiplantibacillus pentosusP1OAXL1022002021222001
1Lactiplantibacillus pentosusP1OAXL2222002021202001
1Lactiplantibacillus
plantarum		P1OAXL3222022202102001
1Lactiplantibacillus pentosusP1OAXL4222002021022001
1Lactiplantibacillus
plantarum		P1OAXL5222000003222001
1Lactiplantibacillus subsp. argentoratensisP1OAXL6222222223222001
1Lactiplantibacillus
plantarum		P1OAXL7222220023002001
1Lactiplantibacillus
plantarum		P1OAXL8222020011101001
1Lactiplantibacillus pentosusP1OAXL9222022011101001
1Bacillus pumilusP1OAXL10222220011001001
2Bacillus amyloliquefaciensP2OAXL11112011024002011
2Enterococcus faecalisP2OAXL14012101221002001
3Lactiplantibacillus sp.P3OAXL15222222021022001
3Staphylococcus sp.P3OAXL16222000021102001
3Enterococcus faecalisP3OAXL17112202202102001
3Bacillus amyloliquefaciensP3OAXL18222022021022001
3Lactiplantibacillus
plantarum		P3OAXL19222122003222001
3Lactiplantibacillus
plantarum		P3OAXL20222122223222001
3Bacillus amyloliquefaciensP3OAXL21012002023002001
4Enterobacter sichuanensisP4OAXL61112021021222001
4Enterobacter sp.P4OAXL62122021003222000
4Lacticaseibacillus sp.P4OAXL64112022023222001
4Lactiplantibacillus diolivoransP4OAXL65211021021222001
4Enterobacter sp.P4OAXL66222022023222000
4Bacillus sp.P4OXL11222202021001111
4Lactiplantibacillus plantarumP4OAXL22222122222222001
4Leuconostoc mesenteroidesP0AXL24222122200112201
4Bacillus amyloliquefaciensP4OAXL25222022001222201
4Lactiplantibacillus plantarumP4OAXL26222122001222201
4Bacillus sp.P4OAXL27222022001222201
4Levillactobacillus brevisP4OAXL28221111221202201
4Enterococcus gallinarumP4OAXL29222120002022201
Group: Corresponds to the Palenque of origin (P1 to P4), GAL: Galactose, GLU: Glucose, FRU: Fructose, MAN: Mannose, LAC: Lactose, SUC: Sucrose. LM: Litmus milk. LBS: Agar Rogosa, M17: Streptococcus lacteus agar, ELLI: Elliker agar, MSE: Mayeux, Sandine & Elliker agar. a Fermentable carbohydrate: (2) positive, (1) weak, (0) negative. b GAS: Gas production: (2) positive, (1) weak, (0) negative. c NO3 Reduction: (2) positive, (1) weak, (0) negative. d LM: Litmus milk reaction: (1) acid clot, (2) chemical reduction with acidification, (3) chemical reduction, (4) alkaline clot. e Media growth in LBS, M17, Elliker or MSE: (2) positive, (1) weak, (0) negative. f Catalase: (1) positive, (0) negative. g Gram value: (1) positive, (0) negative. h The differential staining of bacteria called GRAM: (1) weak, (0) negative.
Table 3. Effect of ethanol concentration (0%, 6%, and 8%) on biomass growth (×106 CFU/mL) and relative population (%) of bacterial strains during fermentation in MRS medium.
Table 3. Effect of ethanol concentration (0%, 6%, and 8%) on biomass growth (×106 CFU/mL) and relative population (%) of bacterial strains during fermentation in MRS medium.
StrainBiomass Growth in MRS Media with Ethanol
(×106 UFC/mL)		Maximum Population Relative
0%6%8%6%8%
P1OAXL1Lactiplantibacillus pentosus40.2014.004.603533
P1OAXL2Lactiplantibacillus pentosus40.1038.0026.809571
P1OAXL3Lactiplantibacillus plantarum40.1012.002.363020
P1OAXL4Lactiplantibacillus pentosus40.4036.0014.028939
P1OAXL5Lactiplantibacillus plantarum38.8034.5026.608977
P1OAXL6Lactiplantibacillus plantarum subsp. argentoratensis40.5014.202.613518
P1OAXL7Lactiplantibacillus plantarum18.206.700.843713
P1OAXL8Lactiplantibacillus plantarum40.509.944.692547
P1OAXL9Lactiplantibacillus pentosus28.409.676.423466
P1OAXL10Bacillus pumilus27.7011.904.264336
P2OAXL11Bacillus amyloliquefaciens16.107.344.264658
P3OAXL15Lactiplantibacillus sp.28.408.944.663152
P3OAXL18Bacillus amyloliquefaciens16.505.663.163456
P3OAXL19Lactiplantibacillus plantarum40.4039.1032.109782
P3OAXL20Lactiplantibacillus plantarum37.5035.8029.409582
P3OAXL21Bacillus amyloliquefaciens15.204.112.162753
P4OAXL11Bacillus sp.51.1033.3020.306561
P4OAXL64Lacticaseibacillus sp.12.703.862.153056
P4OAXL65Lactiplantibacillus diolivorans19.903.981.482037
Table 4. Etnobiotechnological variables analyzed in four Palenques of Oaxaca.
Table 4. Etnobiotechnological variables analyzed in four Palenques of Oaxaca.
Palenque 1Palenque 2Palenque 3Palenque 4
LocalizationMunicipalitySan Dionisio OcotepecSan Pablo EtlaConcepción
Buenavista		San Pedro
Teozacoalco
DistrictTlacolulaEtlaCoixtlahuacaNochixtlán
RegionValles CentralesValles
Centrales		CañadaMixteca
Location
geographic		North latitude (parallels)16°41′39.84″–16°52′24.24″17°07′21.00″–17°11′15.72″17°51’35.28″–18°06’27.00″16°55’49.44″–17°04’31.80″
Western longitude (meridians)96°25′26.76″–96°11′54.24″96°48′11.52″–96°39′11.88″97°34’58.80″–97°21’46.44″97°19’26.76″–97°13’36.48″
Altitude (m asl)1700–22001500–33001600–23001300–2400
ClimateTypeSemi-dry, semi-warmSemi-warm, sub-humid, and temperate sub-humidSemi-dry, semi-warmSemi-warm, Asub-humid
Average annual temperature (°C)20182022
Average annual precipitation (mm)600–700700–800500–600800–1000
HydrographyBasinTehuantepec riverAtoyac riverHondo riverAtoyac river
Land use and vegetation (% of territory) [41,42]Agricultural area9.9223.030.9723.19
Urban area1.7416.870.71.03
Pine-Oak Forest47.954.442.1659.98
Tropical deciduous forest32.06NF23.791.72
Matorral hierophileNFNF2.11NF
Induced grassland [Reference]8.385.730.2714.08
Population
[41,42,43]		Total number of inhabitants 11,41117,1167521153
Total of women 59989083377578
Total of men 54138033375575
Population growth % (2010–2020) 8.6810.20−12.70−9.83
Native language ZapotecoZapotecoChocholtecoMixteco
The population in a situation of
poverty (%) 		992275.9391.67
Marginalization index HighExtremely lowHighHigh
Social backwardness index HighExtremely lowMediumHigh
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López-Sánchez, C.; Espinoza-Martínez, V.A.; Palma-Cruz, F.d.J.; Enríquez-Valencia, R.; Ramírez-López, M.P.; Ortiz-Cortés, L.Y.; Álvarez-Gutiérrez, P.E. Ethnobiotechnological Analysis of the Lactic Bacterial Diversity in the Mezcal Fermentation of Four Palenques in Oaxaca, Mexico. Fermentation 2026, 12, 89. https://doi.org/10.3390/fermentation12020089

AMA Style

López-Sánchez C, Espinoza-Martínez VA, Palma-Cruz FdJ, Enríquez-Valencia R, Ramírez-López MP, Ortiz-Cortés LY, Álvarez-Gutiérrez PE. Ethnobiotechnological Analysis of the Lactic Bacterial Diversity in the Mezcal Fermentation of Four Palenques in Oaxaca, Mexico. Fermentation. 2026; 12(2):89. https://doi.org/10.3390/fermentation12020089

Chicago/Turabian Style

López-Sánchez, Claudia, Víctor Adrián Espinoza-Martínez, Felipe de Jesús Palma-Cruz, Raúl Enríquez-Valencia, Marcos Pedro Ramírez-López, Lourdes Yaret Ortiz-Cortés, and Peggy Elizabeth Álvarez-Gutiérrez. 2026. "Ethnobiotechnological Analysis of the Lactic Bacterial Diversity in the Mezcal Fermentation of Four Palenques in Oaxaca, Mexico" Fermentation 12, no. 2: 89. https://doi.org/10.3390/fermentation12020089

APA Style

López-Sánchez, C., Espinoza-Martínez, V. A., Palma-Cruz, F. d. J., Enríquez-Valencia, R., Ramírez-López, M. P., Ortiz-Cortés, L. Y., & Álvarez-Gutiérrez, P. E. (2026). Ethnobiotechnological Analysis of the Lactic Bacterial Diversity in the Mezcal Fermentation of Four Palenques in Oaxaca, Mexico. Fermentation, 12(2), 89. https://doi.org/10.3390/fermentation12020089

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