Maize or corn (Zea mays), the “gift of the goddess” of the ancient Amerindians, is a cereal crop that is produced in a wide range of agro-ecological environments worldwide. Archeological evidence from the central Andes indicates that the role of maize changed between A.D. 500 and 1500, shifting from a culinary item, simply prepared by boiling, to a more complex symbolic food with elaborated political meanings, transformed by grinding and chewing into beer (Hastorf and Johannessen, 1993). Today maize plays an extremely important role in the Andean culture and is the main cereal grain in Colombia, as measured by production of 260.700 Ha¹ (2015).

Fermented foods, besides being more than a pleasure and a satisfaction of nutritional needs, are a rich source of insight into many aspects of cultural life (Hastorf and Johannessen, 1993). Since pre-Hispanic times, fermented products from maize have been consumed widely in Colombia (Chaves-López et al., 2014a), where their manufacturing remains a traditional art in houses, villages, and small-scale industries. In Colombian fermented foods and beverages, microbial interactions have been demonstrated to be of paramount importance in the development of the particular traits of the final product (Chaves-López et al., 2014b).

Masa Agria, also known as Maiz añejo, is a traditional fermented maize dough, produced in Colombia, which is still prepared by natural fermentation. In the traditional method of preparation, described by Chaves-López et al. (2014a), yellow maize kernels are peeled, covered with water, and stored in a hot place (35 to 40°C) to favor spontaneous fermentation. The period of fermentation varies from 3 to 5 days and determines the degree of sourness with a final pH from 4.4 to 3.8. After fermentation, the grains are washed with water, drained and milled, and then a dough is formed and allowed to stand for 3 or 5 days. During this period, dehydration occurs, and a_w is reduced at different levels depending on the producers. As reported by the same authors, fermentation is triggered mainly by lactic acid bacteria (LAB) and yeasts that are able to reach values of about 8.8 and 6.8 log CFU/g, respectively. Masa Agria is a very common product in Colombian gastronomy, as it is commonly used to prepare soups, tamales (dough filled with meat and vegetables and cooked in a banana leaf), empanadas (cooked dough filled with meat and potatoes and then fried), carantanta (the leftover material in the bottom of the pot that is peeled off after cooked Masa Agria for empanadas) and envueltos (dough steamed in a corn husk).

In the last years, metagenomic analyses have been broadly used to investigate microbial communities of fermented foods (Sakamoto et al., 2011; Chao et al., 2013; Elizaquível et al., 2015; Minervini et al., 2015). In general, these studies revealed a widely unknown microbial biodiversity, underreported by conventional cultivation-based methods. Although several studies have been conducted to explore the bacterial comunities in fermented maize doughs from different countries, to the best of our knowledge no information is available on the microbial communities of the Colombian Masa Agria. Therefore, the aim of this study is to explore the microbial diversity of Colombian Masa Agria by pyrosequencing of 16S rRNA of the bacterial microbiota, to improve the knowledge on microbial communities that can be essential for product characterisation and process optimization (Oguntoyinbo et al., 2011).

In this preliminar study, we used the pyrosequencing of tagged 16S rRNA gene amplicons to explore the bacterial microbiota in Colombian maize fermented dough Masa Agria. The data here presented provide a detailed insight of the bacterial profile of this product, as in our knowledge this is the first report of the bacterial community and structure of this Colombian fermented dough.

Microbial culture revealed growth on the plate counts media used. While the presence of presumptive Lactococcus, Lactobacillus, acetic bacteria and Pseudomonas was confirmed by 16S rRNA pyrosequencing, micro-staphylococci and enterococci were not confirmed. Enterococci may derive from raw materials, such as maize kernels or water, from the environment, and also from the tools used during grain milling and dough manufacturing (Elizaquível et al., 2015). Being able to grow over a wide range of temperature and easily supporting acid pH (Giraffa, 2002; Serio et al., 2010), their presence in the fermented Masa Agria would be unsurprising. Nevertheless our Masa Agria samples were market samples, analyzed at the end of the fermentations process, therefore enterococci deriving from raw materials and environment could have been overgrown by LAB species, as it happens in sourdough, where they are usually found in the first days (De Vuyst et al., 2014). Some authors reported that Slanetz and Bartley Agar lacks of selectivity when used for food samples, as it could over-estimate the actual number of enterococci, being able to support the growth also of lactococci and some Lactobacillus strains (Klein and Reuter, 2012). On the other side, also Mannitol Salt Agar was demonstrated to give false positive results as regards staphylococci, therefore lacking of specificity (Kircher et al., 2002). Pseudomonas spp. were present at extremely low sequence reads; indicating that the counts of bacteria that grew on the medium used (PSB + CFC) were unlikely to be all Pseudomonas. In fact, although the used medium is selective for this genus, the growth of other Gram-negative bacteria such as Serratia marcescens, Aeromonas spp., Xanthomonas spp., Alcaligenes spp., and Acinetobacter spp. is possible (Krueger and Sheikh, 1987).

The presence of lactobacilli, acetic bacteria and yeasts explains the low pH values profiles of the analized doughs from six different producers. The pH values observed in Masa Agria samples, are compatible with those reported in literature for other maize doughs (Annan et al., 2003). Chaves-López et al. (2014b) reported the presence of lactic and acetic acids in Masa Agria samples obtained from the same geographic area in Colombia. These acids not only are responsible for the sour taste, but their effect in pH reduction play a key role in the activation of endogenous and bacterial phytases, increasing dough nutritional value. In fact, phytic acid complexes amino acids and minerals, therefore acting as anti-nutritional factor. High LAB abundance and low pH are also responsible for coliforms decrease (Elizaquível et al., 2015).

Our findings showed that complex microbiota is associated to natural fermented maize doughs and that community membership and structure considerably differed depending on the producer. In fact, some OTUs were detected only in samples from one producer; in addition, the bacterial composition changed in terms either of species and of their relative abundance. This is not surprising, because the bacterial composition of fermented doughs differed on the bases of ecological parameters such as type of maize used, temperature, time of fermentation, water activity, etc. Another possible explanation for differences among the samples could derive from phytochemical treatments that the maize was subjected to, during the cultivation. In fact, the total microbial population and the relative species proportion on maize grains can be affected by many factors, mainly temperature and rainfall, physical damage due to insects and application of phytopharmaceuticals. Although the producers of the analyzed samples of Masa Agria in this study did not know the provenience of the maize, in Colombia it is cultivated from 350 to 2400 meters above sea level. Thus it is evident that bacteria from this natural environment are different and their growth during the fermentation process lead to particular treats of each Masa Agria.

Interestingly, 4% of the readings could not be associated with a known species suggesing that Masa Agria may be an unexplored reservoir of unknown bacterial species.

In different Masa Agria many bacterial species have been found, and some of them have been commonly reported in indigenous Mexican or African fermented maize dough, considerably contributing to the development of the final characteristics of the products (Ben-Omar and Ampe, 2000; Escalante et al., 2001; Abriouel et al., 2006; Assohoun-Djenia et al., 2016). However, the predominant bacterial consortium depends on its source of production with mixtures of maltose and non-maltose fermenting species. As regards the bacterial active population in Masa Agria, evidenced by 16S rRNA pyrosequencing, it is immediately clear that LAB, and particularly Lactobacillus spp. and Weissella spp., together with Acetobacter spp., were dominant in all the samples. In particular, while several differences were observed among samples from the different producers, the two species L. plantarum and A. fabarum were common in all the analyzed doughs. The sequences obtained in the samples from the different producers exhibited high similarities and the species L. fermentum, L. vaccinostercus, P. argentinicus, and Sphingobium spp. were repeatedly found. This observation suggests that these particular species could play a specific role in Masa Agria production and that they could be a typical microbiota of this type of maize fermented product, although the variability should be deeply investigated.

In Mexican fermented maize dough Pozol, Escalante et al. (2001) reported the dominance of Lc. lactis followed by L. alimentarius, L. plantarum, and Streptococcus suis. In addition, Ben-Omar and Ampe (2000), studying the bacterial succession during its production, at the end of the fermentation detected L. plantarum, L. fermentum, L. casei, S. bovis, Bifidobacterium minimum, or Exiguobacterium aurantiacum. Halm et al. (1993) concluded that a homogenous group of obligatively heterofermentative lactobacilli related to L. fermentum and L. reuteri played a dominating role during the production of Ghanaian maize dough Kenkey. On the other hand, the analysis of bacterial community composition of maize dough samples from the Congo Republic, by 16S rRNA gene temporal temperature gradient gel electrophoresis (TTGE), revealed that the most intense band corresponded to L. plantarum/paraplantarum; moreover, although other bacteria such as L. gasseri, Enterococcus spp., L. delbrueckii, L. reuteri, L. casei, L. acidophilus, L. delbrueckii, E. coli, and Bacillus spp. were detected, they were represented by DNA bands of lower intensities (Abriouel et al., 2006). Recently Assohoun-Djenia et al. (2016) reported the predominance of L. fermentum, L. plantarum, P. pentosaceus, P. acidilactici, and W. cibaria species in Doklu from Côte d’Ivoire.

The selective pressure of tropical environments may favor microbial biodiversity and highlights a useful technological potential (Chaves-López et al., 2009), and the geographical isolation among the fermented maize dough products leads to great divergent microbial communities, agreeing with the fact that each maize fermented dough can be considered as unique. Nevertheless it is evident that L. plantarum and L. fermentum are important species in fermented maize dough. Kunene et al. (2000) suggested that L. plantarum and L. fermentum associate in spontaneous fermentation of cereals-based foods.

The importance of L. fermentum in maize fermentation has been confirmed by previous researches in Ghanan, Benin, Mexican fermented maiz doughs (Agati et al., 1998; Ampe et al., 1999; Hayford et al., 1999; Oguntoyinbo et al., 2011; Obinna-Echem et al., 2014) and the high amylolitic activities found in different strains suggest that L. fermentum may be a key organism for fermentation of maize, making the large amounts of starch available to the overall community. In addition, the fermentation products (lactate, formate, and ethanol) may also serve as carbon sources for organisms, such as yeasts (Ben-Omar and Ampe, 2000). Interestingly, Assohoun-Djenia et al. (2016) reported a high prevalence of bacteriocin-producing L. fermentum strains, and their detection in different stages of Doklu production indicates a high potential of these strains to grow and dominate the microbial population in the fermented maize dough.

The amylolytic activity of some strains of L. plantarum, L. lactis, Streptococcus spp. and Leuconostoc mesenteroides had been also reported (Sanni et al., 2002; Díaz-Ruiz et al., 2003). In particular the presence of amylolytic L. plantarum in cereal fermented products is associated to (i) increasing the availability of energy sources for other associated non-amylolytic LAB (ii) contributing to a rapid pH decrease, and (iii) imparting favorable rheological properties to the dough (Sanni et al., 2002). L. plantarum is considered a highly acid-tolerant LAB that dominates in fermentation processes with vegetables and cereals, due to its metabolic flexibility and low pH adaptation (Vrancken et al., 2011). Also several strains of this species have been reported to display a broad spectrum of anti-fungal activity (Schnürer and Magnusson, 2005). Thus, it is possible that these features (amylolytic activity, acid tolerance, and bacteriocin production) contributed to the consolidated presence of both L. plantarum and L. fermentum in maize fermented dough.

Acetobacter, which was the second most represented genus in Masa Agria, varied in abundance among the samples, and was particularly abundant in those from producers 3, 4, and 5. Members of this genera are obligately aerobic bacteria that oxidize ethanol to acetic acid, although some species are also able to further oxidize acetic acid completely to CO₂ and water (Hutkins, 2006). Different species of the genus Acetobacter have been associated with whole crop maize silage (Oude Elferinck et al., 2001), where they dominate the first step of fermentation (Sträuber et al., 2012), together with LAB. While the presence of acetic acid bacteria in naturally fermented wheat dough, such as sourdough, is uncommon, on the contrary Gluconobacter oxydans and A. xylinum, together with L. saccharolyticum and Saccharomyces cerevisiae have been demonstrated to be important in the fermentation of sorghum grains to produce Hussuwa (El Nour et al., 1999). Acetobacter genus has been also associated with maize doughs (Ampe et al., 1999).

Dough fermentation leads to selective environmental conditions, due to sugar consumption and to the progressive pH reduction. Being acid-tolerant, and utilizing also molecules other than sugars for their energetic needs, Acetobacter spp. could be selected in the later stage of fermentation. In a polyphasic study on spatial distribution of microorganisms in Pozol from Mexico, Ampe et al. (1999) reported the presence of yeasts, fungi, EPS producers (including members of the genus Leuconostoc), and enterobacteria, as well as other non LAB, such as members of the genus Acetobacter, at the periphery of a pozol ball, in the outer part. Thus it can be hypothesized that samples of producers 3, 4, and 5 were collected overall from the surface of the dough, where oxygen should not have been a limiting factor for the growth of this genus, while the low presence found in samples from producers 1, 2, and 6 could probably be related to the poor presence of Acetobacter inside the dough. Further studies on spatial distribution of microorganisms in Masa Agria should be performed to confirm this hypothesis.

The presence of Proteobacteria observed in Masa Agria samples, such as Comamonas, Sphingomonas, Acinetobacter, and Pseudomonas spp., has been also recognized in the first step of rye and durum wheat sourdough fermentation (Ercolini et al., 2013), as flour and environment contaminants. In sourdough these genera usually become dominated by LAB such as Lactobacillus and Weissella in the following fermentation steps. However, it has to be underlined that Masa Agria production does not imply refreshments as in the case of sourdough, but the fermentation first of maize kernels and then of maize dough, therefore determining different dynamics of microbial succession.

Girma et al. (1989) observed that Pseudomonas aeruginosa inoculated in the Ethiopian fermented bread Tef injera, grew well until dough pH was reduced to 5.5, and thereafter the population decreased until only few viable cells were isolated at pH 4.0. Nevertheless, Pseudomonas species are characterized by a wide metabolic adaptability to substrates and stressing conditions, thus peculiar species could be selected by the Masa Agria environment. On the other hand, Enterobacteriaceae such as Escherichia, Serratia spp., and Enterobacter aerogenes which could derive from the maize kernels, but more probably from the water used for the dough, were only recognized in samples from producers 2 and 6. It is noteworthy the absence of enteropathogenic species.

Several species of environmental origin were recognized in the different samples and particularly notable are Sphingobium spp. and Dechloromonas spp., playing a role in soil and water bioremediation (Young et al., 2007), and Gemmata spp. (sample 5), a freshwater bacteria originally isolated from Queensland. The presence of Sphingobium spp. is correlated with the water used to wet the maize during Masa Agria production, as it has the capacity to survive in chlorinated waters, allegedly due to the oligotrophic character and the production of biofilms (Vaz-Moreira et al., 2011). This species has been involved in the degradation of chloroacetamide herbicide butachlor (Kim et al., 2013). On the other hand, the relative abundance of Phytoplasma, a plant pathogen able to cause maize bushy stunt that is among the most widespread diseases in herbaceous hosts, causing severe yield losses, may be assumed by the fact that the dough samples from producers 1, 2, and 6 were presumptively obtained from maize of low quality.

The complex microbiota of Masa Agria included also some less-abundant species such as some Lactobacillus (curvatus, rossiae, and silingis), and L. citrineum that migh play an important role in effectiveness and stability of the microbial community, as their microbial metabolism provides molecules able to affect this food ecosystem.

The combined application of culture-dependent and culture-independent analytical strategies allowed us to obtain an insight on the richness of the microbiota of Masa Agria, providing information on the diversity and on the relative abundance of microbial species.

For the first time, this study explored the microbial diversity of Masa Agria, by pyrosequencing of 16S rRNA gene amplicons. Our results elucidated the structures of the bacterial communities of six samples obtained from different producers, identified specific dominant species, and suggested the presence of possibly unknown microorganisms.

In particular, this research was focused on bacterial characterisation at the end of fermentation in commercial samples. Further investigations are needed to evaluate the microbial dynamics throughout the manufacturing process and to investigate the role of the different bacterial groups during fermentation.

CL: Devised and drafted the manuscript-statistical analyses. AS: drafted the manuscript. JO: molecular analysis. CR: culture dependent analyses. CT: statistical analyses. AP: manuscript revision.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.