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The metaproteomic approach allows a deep microbiome characterization in different complex systems. Based on metaproteome data, microbial communities' composition, succession, and functional role in different environmental conditions can be established.The main challenge in metaproteomic studies is protein extraction, and although many protocols have been developed, a few are focused on the protein extraction of fermented foods. In this chapter, a reproducible and efficient method for the extraction of proteins from a traditionally fermented starchy food is described. The method can be applied to any fermented food and aims to enrich the extraction of proteins from microorganisms for their subsequent characterization.

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Pozol is a traditional prehispanic Mexican beverage made from fermented nixtamal dough; it is still part of everyday life in many communities due to its nutritional properties. It is the product of spontaneous fermentation and has a complex microbiota composed primarily of lactic acid bacteria (LAB). Although this is a beverage that has been used for centuries, the microbial processes that participate in this fermented beverage are not well understood. We fermented corn dough to produce pozol and sampled it at four key times to follow the community and metabolic changes (0, 9 24 and 48 h) by shotgun metagenomic sequencing to determine structural changes in the bacterial community, as well as metabolic genes used for substrate fermentation, nutritional properties and product safety. We found a core of 25 abundant genera throughout the 4 key fermentation times, with the genus Streptococcus being the most prevalent throughout fermentation. We also performed an analysis focused on metagenomic assembled genomes (MAGs) to identify species from the most abundant genera. Genes involving starch, plant cell wall (PCW), fructan and sucrose degradation were found throughout fermentation and in MAGs, indicating the metabolic potential of the pozol microbiota to degrade these carbohydrates. Complete metabolic modules responsible for amino acid and vitamin biosynthesis increased considerably during fermentation, and were also found to be abundant in MAG, highlighting the bacterial contribution to the well-known nutritional properties attributed to pozol. Further, clusters of genes containing CAZymes (CGCs) and essential amino acids and vitamins were found in the reconstructed MAGs for abundant species in pozol. The results of this study contribute to our understanding of the metabolic role of micro-organisms in the transformation of corn to produce this traditional beverage and their contribution to the nutritional impact that pozol has had for centuries in the traditional cuisine of southeast Mexico.

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Pozol is a Mexican beverage prepared from fermented nixtamalized maize dough. To contribute to understanding its complex microbial ecology, the effect of inoculating on MRS-starch pure and mixed cultures of amylolytic Sii-25124 and non-amylolytic W. confusa 17, isolated from pozol, were studied on their interactions and fermentation parameters. These were compared with L. plantarum A6, an amylolytic strain isolated from cassava. Microbial growth, kinetic parameters, amylolytic activity, lactic acid production, and hydrolysis products from starch fermentation were measured. The population dynamics were followed by qPCR. L. plantarum A6 showed higher enzymatic activity, lactic acid, biomass production, and kinetic parameters than pozol LAB in pure cultures. Mixed culture of each pozol LAB with L. plantarum A6 showed a significant decrease in amylolytic activity, lactic acid yield, specific growth rate, and specific rate of amylase production. The interaction between Sii-25124 and W. confusa 17 increased the global maximum specific growth rate (µ), the lactic acid yield from starch (Ylac/s), lactic acid yield from biomass (Ylac/x), and specific rate of lactic acid production (qlac) by 15, 30, 30, and 40%, respectively, compared with the pure culture of Sii-25124. Interactions between the two strains are essential for this fermentation.

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Pozol is an acidic, refreshing, and non-alcoholic traditional Mayan beverage made with nixtamalized corn dough that is fermented spontaneously. The extensive analysis of the microbiology, biochemistry and metaproteomics of pozol allowed the construction of a comprehensive image of the fermentation system. The main changes in both the substrate and the microbiota occurred in the first 9 h of fermentation. The increase in microorganisms correlated with the drop in pH and with the decrease in the contents of carbohydrates, lipids, and nitrogen, which shows that this stage has the highest metabolic activity. Bacterial proteins were mainly represented by those of lactic acid bacteria, and among them, the proteins from genus Streptococcus was overwhelmingly the most abundant. Yeast proteins were present in all the analyzed samples, while proteins from filamentous fungi increased up to 48 h. The metaproteomic approach allowed us to identify several previously unknown enzyme complexes in the system. Additionally, enzymes for hydrolysis of starch, hemicellulose and cellulose were found, indicating that all these substrates can be used as a carbon source by the microbiota. Finally, enzymes related to the production of essential intermediates involved in the synthesis of organic acids, acetoin, butanediol, fatty acids and amino acids important for the generation of compounds that contribute to the sensorial quality of pozol, were found.

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[This corrects the article DOI: 10.3389/fmicb.2021.629449.].

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The genus Weissella is composed of a group of Gram-positive facultative anaerobe bacteria with fermentative metabolism. Strains of this genus have been isolated from various ecological niches, including a wide variety of fermented cereal foods. The present study aimed to determine the relative abundance and fermentation capabilities of Weissella species isolated from pozol, a traditional maya product made of lime-cooked (nixtamalized) fermented maize. We sequenced the V3-V4 regions of 16S rDNA; Weissella was detected early in the fermentation process and reached its highest relative abundance (3.89%) after 3 h of culture. In addition, we evaluated five Weissella strains previously isolated from pozol but reported as non-amylolytic, to define alternative carbon sources such as xylan, xylooligosaccharides, and sucrose. While no growth was observed on birch xylan, growth did occur on xylooligosaccharides and sucrose. Strains WcL17 and WCP-3A were selected for genomic sequencing, as the former shows efficient growth on xylooligosaccharides and the latter displays high glycosyltransferase (GTF) activity. Genomes of both strains were assembled and recorded, with a total of 2.3 Mb in 30 contigs for WcL17 and 2.2 Mb in 45 contigs for WCP-3a. Both strains were taxonomically assigned to Weissella confusa and genomic analyses were performed to evaluate the gene products encoding active carbohydrate enzymes (CAZy). Both strains have the gene content needed to metabolize sucrose, hemicellulose, cellulose, and starch residues, all available in pozol. Our results suggest that the range of secondary enzymatic activity in Weissella confusa strains confer them with wide capabilities to participate in fermentative processes of natural products with heterogeneous carbon sources.

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Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

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Streptococcus infantarius ssp. infantarius 25124 (Sii-25124) is a lactic acid bacterium (LAB) isolated from pozol, a refreshing beverage prepared by suspending fermented nixtamal (a thermal and alkali-treated maize dough) in water. Although Lactobacillus are the predominant strains in fermented doughs, such as sourdoughs, and non-nixtamalized fermented maize foods, the pozol microbiota is markedly different. This may be the result of the nixtamalization process, which could act as a selective force of some strains. Sii-25124 has been reported as the main amylolytic LAB in pozol; starch is the primary carbon source on nixtamal since monosaccharides and disaccharides are lost during nixtamalization; however, non-amylolytic LAB counts are higher than amylolytic LAB in pozol after 24-h fermentation suggesting that another carbon source is being used by the former bacteria. Hemicellulose (arabinoxylan in maize) becomes available via nixtamalization and is subsequently metabolized by LAB. The aim of this work was to determine whether this bacterium is able to use arabinoxylan as the only carbon source in a defined medium containing arabinoxylan extracted from either nejayote (wash water produced during nixtamal preparation), or beechwood xylan. Xylanase activity in the presence of nejayote arabinoxylan (135.8 ± 48.7 IU/mg protein) was higher than that of beechwood (62.5 ± 19.8 IU/mg protein). Other enzymatic activities, such as arabinofuranosidase and acetyl esterase, were also detected, suggesting the adaptation of the bacterium studied to nixtamal dough. It was concluded that Streptococcus infantarius 25124 isolated from pozol was able to use arabinoxylans, which are present in nixtamal dough, so fermentation does not depend exclusively on free sugars and starch.

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Pozol is a traditional fermented maize dough prepared in southeastern Mexico. Wide varieties of microorganisms have already been isolated from this spontaneously fermented product; and include fungi, yeasts, and lactic- and non-lactic acid bacteria. Pozol presents physicochemical features different from that of other food fermentation products, such as a high starch content, in addition to a low protein content. It is these qualities that make it intractable for protein recovery and characterization. The aim of this study was to develop a methodology to optimize the recovery of proteins from the pozol dough following fermentation, by reducing the complexity of the mixture prior to 2D-PAGE analysis and sequencing, to allow the characterization of the metaproteome of the dough. The proteome of 15day fermented maize dough was characterized; proteins were separated and analyzed by mass spectrometry (LC-MS/MS). Subsequent sequence homology database searching, identified numerous bacterial and fungi proteins; with a predominance of lactic acid bacterial proteins, mainly from the Lactobacillus genus. Fungi are mainly represented by Aspergillus. For dominant genera, the most prevalent proteins belong to carbohydrate metabolism and energy production, which suggest that at 15days of fermentation not only fungi but also bacteria are metabolically active. BIOLOGICAL SIGNIFICANCE: Several methodologies have been employed to study pozol, with a specific focus toward the identification of the microbiota of this fermented maize dough, using both traditional cultivation techniques and culture independent molecular techniques. However to date, the dynamics of this complex fermentation is not well understood. With the purpose to gain further insight into the nature of the fermentation, we used proteomic technologies to identify the origin of proteins and enzymes that facilitate substrate utilization and ultimately the development of the microbiota and fermentation. In this paper we overcome the first general challenge for such studies, obtaining a protein sample with adequate quality capable of representing the system.

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