Tejuino, a Traditional Fermented Beverage: Composition, Safety Quality, and Microbial Identification

This study aims to analyze the chemical and microbial composition and characterize volatile compounds from the artisanal and commercial Tejuino beverage. For this, eight samples are analyzed (four artisanal and four commercial). The chemical and microbiological quality is determined by standard methods, and volatile compounds are determined by solid-phase microextraction. Overall, the physicochemical composition and microbiological quality are higher for artisanal Tejuino (p < 0.05). The pH values were 3.20 and 3.62, and 0.76 and 0.46 meq of lactic acid for artisanal and commercial Tejuino, respectively. With volatile compounds analyzed, esters, benzenes, and aldehydes were predominant; meanwhile, ethanol was a volatile compound with the highest concentration for all samples. Saccharomyces cerevisiae and Limosilactobacillus fermentum were identified in artisanal Tejuino; yeasts of the Pichia genera and Lactiplantibacillus plantarum, for commercial Tejuino, and Enterococcus genus were identified in both samples. The characterization of both types of Tejuino allows us to update the information available on this important Mexican beverage. In addition, the isolation of lactic acid bacteria, as representative bacteria of both drinks, offers an area of opportunity to know the potential functionality of these bacteria in traditional fermented products.


Introduction
Corn is characterized by its high content of carbohydrates, protein, dietary fiber, B vitamins, and minerals, so it is considered that its consumption has beneficial effects on health [1]. This product, when is fermented by microorganisms and the presence of some endogenous enzymes (e.g., amylases, proteases, and phytases) reducing their carbohydrate content and improves the bioavailability of B vitamins [2]. Specifically, in traditional corn-based fermented beverages, a great diversity of lactic acid bacteria (LAB), molds, and yeasts have been found to utilize corn nutrients, improving the nutritional properties of the product. For example, LAB increases the free amino acids content, B complex vitamin, minerals (calcium, iron, and zinc), volatile compounds that contribute to the organoleptic characteristics of maize-based fermented beverages [3].
Tejuino is a traditional non-distilled fermented beverage from Mexican produced with germinated or nixtamalized maize. Currently, this beverage is produced and consumed mainly in the western and north-western states of Mexico (e.g., Nayarit, Jalisco, Zacatecas,

Collection of Samples
Samples of commercial Tejuino were purchased at local mobile stores and artisanal Tejuino from Wixárika ethnic groups from Nayarit, Mexico. Samples were collected in two seasons (winter and spring) in different regions of Nayarit. Samples from artisanal Tejuino were codified as "AR" (El Nayar), "AY" (La Yesca), "AW" (Wirikuta), and "TZ" (Zitacua); commercial Tejuino samples were codified as "TC" (City center), "TL" (Leon Street), "TM" (Plaza de la Música), and "TT" (Tecnológico Zone), obtaining a total of eight different beverages. All samples were transported under refrigeration conditions to the laboratory for further analysis.
A fresh sample (1 L) was used for physicochemical analysis (pH, acidity, moisture, total solids) of volatile compounds; for the microbiological analysis, 200 mL of sample was aseptically obtained and placed in sterile flasks and immediately analyzed. On the other hand, 800 mL was frozen at −80 • C and lyophilized (FreeZone 6, Labconco, Kansas City, USA) to determine fat, protein, and ash.

Microbiological Analysis
The microbiology quality was determined following the methodology reported by Torres-Llanez et al. [14]. For this, 10 mL of each Tejuino sample was mixed with 90 mL of peptone water at room temperature and series of 10-fold dilutions were performed. Total Coliform Bacteria (TCB) was evaluated by the most probable number (MPN) method in Lauryl Sulfate Broth (BD Difco, Sparks, MD, USA), and incubated at 25 • C for 48 h. Aerobic mesophilic bacteria (AMB) in Plate Count Agar (BD Difco) at 35 • C for 48 h; molds and yeasts (MY) in Potato Dextrose Agar (BD Difco) acidified at pH 3.5 with tartaric acid solution (10% w/v) (FAGALAB, Sinaloa, Mexico) at 25 • C for 5 days. The cell count was reported as log 10 colony-forming units (CFU)/mL. All analyses were performed following the specifications of the Official Mexican Standard (NOM) [15][16][17].
On the other hand, LAB concentration of Streptococcus spp and Lactococcus spp was determined in M17 agar (BD Difco) supplemented with lactose and dextrose (5%) and incubated at 42 • C and 30 • C for 48 h, respectively; Lactobacillus spp. in de Man, Rogosa and Sharpe agar (MRS BD Difco) at 37 • C for 48 h under anaerobic conditions (BD GasPak Anaerobe Container System, MD, USA). The cell concentration was reported as log 10 CFU/mL.

Isolation of Lactic Acid Bacteria and Yeast
A minimum of two representative LAB colonies was obtained for each medium selected (M17 and MRS agar). Three culture steps in agar-broth-agar were performed, and colony morphology characteristics such as shape, margin, and elevation were considered [18]. Later, the bacteria and yeasts were grown in a specific medium for 18 h. A catalase and oxidase test by Kovacs method and Gram staining were performed. The isolated strains were visualized by microscopy (100 × objective, Modelo AXIO, ZEISS Corporation, Germany). Finally, the strains were stored at −80 • C using 80% glycerol (v/v) [19].

Identification of LAB and Yeasts Strains
LAB identification; each strain was cultured for 18 h in MRS (anaerobic conditions), M17 broth at 37 • C, 30 • C, and 40 • C. Then, 1 mL of each strain was centrifuged (16,000× g, 10 min) (Spectrafuge 16M, Labnet, Woodbridge, NJ, USA) to obtain a pellet and the DNA extraction was performed using the commercial protocol kit PrepMan Ultra Sample Preparation Reagent (Applied Biosystems, Foster City, CA, USA). The DNA purity was determinate at 260 nm (NanoDrop 2000c UV-Vis, Thermo Fisher Scientific, Wilmington, DE, USA), and the DNA integrity was evaluated in agarose gel (Sigma-Aldrich, MO, USA) electrophoresis (80 V, 30 min). It was visualized in a photodocumenter (Gel DocTM XR+System, Bio-Rad, CA, USA). The analysis of the 16S rRNA method was performed following the protocol of MicroSEQ TM 500 identification Systems (Applied Biosystems). The PCR conditions for amplification was performed in a thermocycler endpoint (Eppendorf, Hamburg, Germany). The PCR conditions for denaturation were at at 95 • C for 10 min, 30 cycles of 95 • C for 30 s, 60 • C for 30 s, and 72 • C for 45 s, final extension of 72 • C for 10 min and final step at 4 • C. For the sequencing, 25 cycles were performed at 96 • C for 10 s, 50 • C for 5 s, and 60 • C for 4 min.
For the identification of yeast, this was grown in PDA broth at 25 • C, 18 h. Subsequently, 1 mL of culture was centrifuged (16,000× g, 10 min) (Spectrafuge 16M) for the extraction of DNA using the commercial protocol Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). The amplification method was based on the MicroSEQ TM D2 rDNA Fungal Sequencing Kit (Applied Biosystems). The PCR conditions for amplification initial step was at 95 • C for 10 min, 35 cycles at 95 • C for 30 s, 53 • C for 30 s, 72 • C for 1 min, final extension 72 • C for 10 min. For the sequencing run, 25 cycles were performed at 96 • C for 10 s, 50 • C for 5 s, and 60 • C for 4 min.
Each PCR product was purified with ExoSAP-IT TM PCR Product Cleanup Reagent (Affymetrix, Santa Clara, CA, USA) and, then, sequencing reactions were purified using the BigDye Xterminator TM purification kit (Applied Biosystems) and, the purified sequencing reactions were examined in a 3500 Genetic Analyzer (Applied Biosystems). The sequences obtained were analyzed in the MicroSEQ software v2.0 (Applied Biosystems) and compared with the library of GenBank database (Accessed 1 January 2021; http://www.ncbi.nlm.nih. gov/BLAST/) using the Blast program of NCBI to determinate the identity of the strains. Sequences with a percentage identity > 99% were considered to belong to the same species.

Analysis of Volatile Compounds by Solid-Phase Microextraction Gas Chromatography Mass Spectrometry (SPME-GC-MS)
Headspace solid-phase microextraction and gas chromatography (HS-SPME-GC) method were used following the methodology reported by Reyes-Díaz et al. [20]. For this, fresh Tejuino samples (5 mL), 2 g of NaCl, and 5 µL of dodecanoic acid methyl ester (internal standard, 1000 ppm in methanol, PolyScience C., Niles, IL, USA) were placed in screw-capped glass vials (20 mL, 22.75 × 75 mm) and sealed with headspace vial caps (18 mm magnetic PTFE/Sil, Agilent Technologies, Basel, Switzerland). Then, vials were placed on the GC Sampler system (GC autosampler 80, Agilent Technologies). Before SPME extraction, samples were allowed to equilibrate at 70 • C (150 rpm, 30 min). Afterward, a 75 µm CAR-PDMS fiber (Supelco Co., Bellefonte, PA, USA) was exposed to the sample headspace while shaking (150 rpm) at 70 • C for 60 min. Then, the fiber was withdrawn into the needle of the SPME device and immediately desorbed at 250 • C for 10 min in splitless mode. Volatile compounds were determined into GC-MS System (Agilent Technologies 7890A/5975C, Santa Clara, CA, USA), and the injector was provided with an inlet liner (78.5 mm × 6.5 mm O.D. × 0.75 mm I.D., Sigma-Aldrich, St. Louis, MO, USA). Helium was used as the carrier gas at 1.5 mL/min. The compounds were separated by a high-polarity DB-WAX column (60 m length, 0.25 mm I.D., 0.25-µm film thickness, Agilent J&W Scientific) with a carrier gas at 1 mL/min. The initial oven temperature was held at 45 • C for 12.5 min, increased to 114 • C (rate of 4 • C/min for 6 min), then to 143 • C (rate of 7 • C/min for 15 min), and, finally, increased to 165 • C (rate of 15 • C/min, for 15 min). Volatile compounds were tentatively identified by comparing their spectra to those from the National Institute of Standards and Technology MS library (Soft. v. 2.0 g., 2011).

Statistical Analysis
All dates were analyzed using one way-ANOVA, ant the differences among samples were compared using the Fisher LSD test with p < 0.05. Principal component analysis (PCA) by factor loadings and factor scores was performed to obtain volatile compounds data; clusters were determined by k-means clustering analysis. All the statistical analyses were performed by using the NCSS-2007 statistical software (Kaysville, UT, USA).

Chemical Composition
The results of the physicochemical composition of the samples of Tejuino are shown in Table 1. Overall, the parameters evaluated showed differences (p < 0.05) among the samples evaluated. In particular, the artisanal samples presented a higher moisture (89.73-92.66%) compared to the commercial samples (83. .29%) and, therefore, the TS were lower for the artisan samples (7.34 to 10.28%). The protein content showed a difference (p < 0.05), being 7.3-fold higher for the artisanal samples than commercial. On the other hand, the fat content was 1.81-fold and ash was 3.10-fold higher for artisanal samples (p < 0.05). On the contrary, the CHOS content was higher for commercial samples with a mean of 78.70%; meanwhile, for artisanal samples, it was 62.48% (p < 0.05). The pH range was 2.91 to 3.52 and 3.36 to 3.96; for acidity there was 0.4 to 1.22 and 0.27 to 0.57 meq of lactic acid/L for the artisanal and commercial beverages, respectively, the artisanal beverages being the ones to show the higher acidity. Although, a difference of (p > 0.05) among artisanal and commercial Tejuino was seen. The elaboration process could explain the difference in the physicochemical composition between artisanal and commercial Tejuino beverages. Corn germination is conducted in dark conditions; then, the sun drying process proceeds (2-3 days), finally being mixed with water. The liquid was cooking and the sediments were fermented for 24 h at room temperature. Afterwards, both samples were mixed, filtered, and the filtered sample was left to ferment for 7 days. On the other hand, commercial Tejuino is produced from nixtamalized corn, followed by the addition of piloncillo to initiate fermentation for 12-24 h [5]. In particular, the combined processes of fermentation and germination improve the nutritional compositions of beverages. A study reported by Hiran et al. [21] showed an enhanced-up to two-fold-protein content of fermented germinated maize seeds by LAB. Furthermore, during germination, endogenous enzymes hydrolyze the maize compounds, e.g., prolamins and glutelins [22].
The CHOS concentration for commercial Tejuino may be influenced by the addition of "piloncillo" (brown sugar, prepared from the undistilled juice of sugar that contains >70% of sugar). On the contrary, the starch present in the germinated corn is hydrolyzed into sugars such as glucose and saccharose [23]. During the fermentation, the microorganisms reduce the carbohydrates content up to 20% [21], as well as the ash content, but enhance the protein content [24].
The values of pH and acidity for artisanal and commercial Tejuino did not show differences, but AR and AY were the lowest. Fermentation is spontaneous and uncontrolled in traditional fermented products; artisanal Tejuino has up to 6 days of fermentation, where several microorganisms and enzymatic processes hydrolyze molecules such as lipids, proteins, and carbohydrates. For instance, Ben Omar and Ampe, [25] associated the pH reduction due to organic products (e.g., lactic acid, formic acid) and ethanol by the action of microorganisms. Although the fermentation time for commercial Tejuino is only 12 to 24 h, the "Tejuino viejo" (starter culture unless 24 h of fermentation), and, finally, some additives are incorporate, such as salt and lemon juice, which may influence the pH values.

Microbial Quality
The microbial quality of Tejuino beverages is shown in Table 2. Overall, the artisanal samples showed the highest concentrations for all types of microorganisms evaluated. Only total coliform bacteria were absent in all samples. All cell concentrations were different (p < 0.05) among samples, but higher for artisanal beverages. The MY range was 7.32 to 8.47 and 4.74 to 7.93 log10 CFU/mL; AMB was 7.27 to 8.66 and 7.17 to 7.59 93 log 10 CFU/mL for artisanal and commercial beverages, respectively. The physicochemical composition of Tejuino determines the microbial diversity. Some conditions of low pH may be a factor that affects the presence of some microorganisms. For instance, LAB predominates in an acidic environment, but there was less or an absence of total enterobacteria [26]. On the contrary, LAB were the predominant group in these beverages. During the nixtamalization processes, the concentration of carbohydrates was reduced, starch being the main carbohydrate available for LAB [27]. This could explain why the commercial beverage had a lower concentration of LAB. Regarding artisanal Tejuino, it showed the highest concentration. This great diversity in the concentration of microorganisms has been reported in other traditional maize-based beverages, such as Pozol [25,27], Atole Agrio [28], and commercial Tejuino [29].
The microbiological quality for both beverages showed marked differences in concentration, which could be attributed to the fact that both processes are non-standardized methods, indicating an absence of good manufacturing practices. However, the presence of microorganisms determines the sensory characteristics of this type of beverage, and even the possible health effects attributed to the great diversity of metabolites present, and even the microbiota present have been documented in other beverages [30]. Moreover, during spontaneous fermentation, bacteria and yeasts produce several metabolites available for other microorganisms, showing a microbial diversity [31].

Identification of LAB and Yeast Strains
LAB and yeast were identified molecularly by comparing the16S rRNA gene sequences with NCBI sequences available in this database (Table 3). Twenty-eight strains were identified with a 100% similarity except for strain number twenty-eight, which showed 95%. The main yeasts identified were Pichia occidentalis and Pichia kudriavzevii for commercial Tejuino, and Saccharomyces cerevisiae for the artisanal Tejuino samples. On the other hand, LAB identification, the predominant bacteria identified were Limosilactobacillus fermentum (five strains) and Lactiplantibacillus plantarum (three strains) for commercial and artisanal Tejuino, Enterococcus durans (five strains) for commercial Tejuino and two strains for artisanal Tejuino, Enterococcus faecium and Staphylococcus warneri (two strains) for commercial, and Lactococcus lactis for artisanal Tejuino.
In this sense, important characteristics were documented for some identified microorganisms, which could be of great interest for future studies. In the yeast group, Pichia genus has been isolated from Chicha (indigenous Andean beer) [32], and has been reported as a start culture [33]. Pichia kudriavzevii has been characterized by the production of biofilms at a low pH [34]; Pichia occidentalis, with the potential to produce phytases and bioethanol in prolonged fermentation times [35,36], also showed a stress tolerance [32]. This yeast has been reported in other fermented beverages such as cachaça, tequila, mezcal, wine, and beer [37]. Saccharomyces cerevisiae has been widely reported for its fermentation properties and ethanol production [32,38], and is present in several traditional fermented beverages of Mexico such as Atole agrio [39], Tesgüino [6], and Tepache [40]. LAB are the dominant microorganism in several foods, but the existence of consortium helps to promote the diversity of microorganisms; LAB produces organic acids that decrease pH and promote the growth of yeast [41]. Furthermore, LAB contributes to food preservation and flavor development by organic acid production. Some reports show increasing digestibility in vitro of the proteins and starch of maize due to LAB consortium by fermentation [42]. However, we found undesirable bacteria such as Staphylococcus, related to food handling practices [43].
During spontaneous fermentation, bacteria and yeasts release several metabolites and promote the growth of other microorganisms. Thus, this interaction enhances the nutritional, rheological, and sensory properties of food [31]. This effect has been documented for other traditional beverages [6,39,40].

Volatile Compounds
The volatile compounds found in the eight samples of artisanal and commercial Tejuino beverages are shown in Table 4. Eighty-nine volatile compounds were identified; however, only ten compounds were detected in all beverages (decanoic acid ethyl ester, acetic acid 2-phenylethyl ester, hexadecanoic acid ethyl ester, ethanol, 1-pentanol, phenylethyl alcohol, octanoic acid, benzaldehyde, creosol, and phenol, 4-ethyl) and showed a difference among artisanal and commercial beverages (p < 0.05) except for cresol and phenol 4ethyl. In this regard, esters, benzenes, and aldehydes were the highest number of volatile compounds identified (26, 17 and 12 compound, respectively). Our data showed the highest relative abundance of volatile compounds for artisanal samples, the esters group being the predominant compounds. The "AR" beverage showed the highest esters; meanwhile, "AW" was for aldehydes and benzenes. Esters may provide fruity and floral flavors in some food; thus, decreasing the astringent aroma of fatty acids and amines [44].   ND  ND  ND  ND  ND  62 3-Acetyl-1H-pyrroline 55.11 0.002 ± 0.00 a 0.008 ± 0.00 b 0.003 ± 0.00 a 0.032 ± 0.02 a ND ND ND Although alcohols were not the predominant group, it is important to highlight that ethanol showed the highest concentration, mainly for artisanal beverages, with 4.17, 3.01, 4.40, and 4.21 ppm, followed by phenyl ethyl alcohol with 1.13, 2.01, 1.6, and 0.9 ppm for "AR", "AW", "AY" and "AZ", respectively. In this sense, the higher alcohol concentration in the artisanal sample could be attributed to the presence of Saccharomyces cerevisiae. This yeast produces enzymes such as amylases, lipases, esterases, and phytases which promote flavor and aroma, and are the most employed for ethanol production, ketones, aldehydes, and esters. Furthermore, the high LAB concentration may favor the enzymes production for hydrolyzing corn starch and the yeasts used for their metabolism, which release vitamins and soluble nitrogen for LAB growth [45]. Other cereal-based fermented co-cultures of LAB and yeast showed a higher alcohol content [46].
Additionally, the analysis of the PCA showed the clustering of volatile compounds and Tejuino sample ( Figure 1). Figure 1a shows the PCA of 89 identified compounds, and three main groups were identified by the k-means cluster analysis and explained with 81.39%, 6.13%, and 5.05% for PC1, PC2, and PC3, respectively. In cluster A found showed the highest concentration of volatile compounds and ethanol was included. In cluster B are compounds that did not show a significant difference; cluster C only contained tree compounds (decanoic acid ethyl ester, hexadecenoic acid ethyl ester, and (E)-9-octadecenoic acid ethyl ester). reported having strong antimicrobial properties against pathogenic microorganisms at low concentrations [59,60]. The relative abundance for aldehydes, only for artisanal Tejuino, may be attributed to soaking that disrupt the physical structure to release volatile compounds and enhance lipoxygenase activity [61]. The fermentative changes directly influence final fermented physical properties, chemical, biological, and sensory properties. This activity is associated with microorganisms, which generate various volatile compounds that promote the development of flavors in fermented foods [62].

Conclusions
Their production process primarily determined the physicochemical characteristics and microbiological quality of the Tejuino beverages. In this aspect, the artisanal beverage was outstanding, and showed a great diversity of volatile compounds. The isolated microorganisms identified were yeasts and lactic acid bacteria, which may be considered for future studies to evaluate the potential bioactivity for their use in industrial processes, as well as an examination of microbiota, which could be carried out to assess its possible potential use in the production of compounds with a biotechnological application. This study allowed us to know and update the available information on this important traditional fermented beverage of Mexico.  The PCA for Tejuino samples is shown in Figure 1b. This analysis showed three components that explained the 35.96%, 27.94%, and 15.00% for PC1, PC2, and PC3, respectively. Commercial beverages (TC, TL, TM, and TT) were grouped at the same cluster. However, artisanal beverages (AR, AW, AY, and AZ) showed a high dispersion, indicating a higher variability among them, considering the distribution of the identified volatile compounds.
Volatile compounds such as esters derived from fatty acids detected in this study could promote fruity notes and have been related to the presence of yeast strains such as Saccharomyces cerevisiae [47][48][49]. Thus, the esters found in Tejuino samples could be attributed to their content of alcohols, which are precursors of these compounds [50].
On the other hand, the content of ethanol was associated with Saccharomyces cerevisiae, which is the most commonly employed yeast for ethanol production [51,52], and this was identified for the artisanal samples of our study. Moreover, yeast produces longchain and complex alcohols that, together with esters, have an interesting flavor, such as phenylethanol, which possesses a rose-like aroma [53,54], as well as sweet alcohol, rough, and fruity aromas [55].
On the other hand, D-limonene (terpene) was reported in both samples and has been associated with a lemon-like [56], flowery, and lilac odor [57,58]. The compounds and β-myrcene were reported in others beverages composed of maize [58]. Otherwise, aldehydes were found in both types of Tejuino. For instance, hexanal has been identified in other cereal-based fermented products and sorghum malt beverages, and has been reported having strong antimicrobial properties against pathogenic microorganisms at low concentrations [59,60].
The relative abundance for aldehydes, only for artisanal Tejuino, may be attributed to soaking that disrupt the physical structure to release volatile compounds and enhance lipoxygenase activity [61]. The fermentative changes directly influence final fermented physical properties, chemical, biological, and sensory properties. This activity is associated with microorganisms, which generate various volatile compounds that promote the development of flavors in fermented foods [62].

Conclusions
Their production process primarily determined the physicochemical characteristics and microbiological quality of the Tejuino beverages. In this aspect, the artisanal beverage was outstanding, and showed a great diversity of volatile compounds. The isolated microorganisms identified were yeasts and lactic acid bacteria, which may be considered for future studies to evaluate the potential bioactivity for their use in industrial processes, as well as an examination of microbiota, which could be carried out to assess its possible potential use in the production of compounds with a biotechnological application.
This study allowed us to know and update the available information on this important traditional fermented beverage of Mexico.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.