Characterization of the Food Microbiota in Ready-to- Eat Mexican Foods

Ready-to-eat food microbiota are the microorganisms present in the dishes that are currently consumed during meals. These microorganisms include those that may have a health benefit, are potentially pathogenic or have not yet been given a function. Foods suitable for consumption are not free of microorganisms; however, within the food industry only yeasts have been given a beneficial function, while other microorganisms such as filamentous fungi and bacteria have been studied for their negative effects on food. We determined the bacterial diversity in samples of highly demanded, freshly prepared, unspoiled ready-to-eat dishes by high-throughput DNA sequencing of 16S rDNA libraries. We found a great bacterial diversity, whereby the most abundant bacterial phyla were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, TM7 and Thermi, among others. These phyla included bacteria with remarkable abundances in some dishes. The alfa diversity analyses showed that the main dishes had the largest diversity. The beta-diversity analyses clustered the bacterial communities of soups, side plates, desserts, and beverages, and some main dishes. Based on our results we conclude that unspoiled ready-to-eat Mexican dishes contain a rich bacterial diversity, which may contribute to the organoleptic properties of the dishes without representing a sanitary risk for the consumers.


Introduction
Foods that are suitable for human consumption are not free of microorganisms. The dishes that we consume regularly, in addition to containing nutrients that contribute to our health, are full of microorganisms contributed by each ingredient [1]. Ready-to-eat food microbiota are the microorganisms present in the dishes that are currently consumed during meals. These microorganisms include those that may have a health benefit, such as probiotics, or may be potentially pathogenic, in addition to others that have not yet been described or given a function [2]. Microbial contamination in food has been broadly described, and includes pathogenic microorganisms, causing food-related diseases, food spoilage or deterioration of the original food properties [3]. There are those that may have a beneficial function for the consumer, such as probiotics, or even some that add desirable organoleptic properties to the food by ageing. Cheeses are an example of ingredients in the recipes of some dishes that contribute to the microbiota present in ready-to-eat food, ingested almost three times every day by the commensals [4]. Although the microbiota present in the food ingredients are usually well characterized [5], the reports about the characterization of the microbiota present in ready-to-eat food are scarce [1]. In this work we explored the microbiota present in ready-to-eat Mexican dishes with the purpose of characterizing the bacterial communities which are ingested in everyday feeding.

Ready-to-Eat Food Sampling
A selection of approximately 35 different dishes with high consumer demand, available in the San-Pedro-Zacatenco area, in northern Mexico City (19.5070° N, 99.1214° W), were aseptically collected and transported immediately to the laboratory to be processed within 30 min for analysis ( Table 1).

DNA Extraction
For DNA extraction, 100 g of each solid dish was homogenized with 100 mL of deionized water using a food hand blender (Braun Cat.# MQ725) in a 500 mL glass beaker. An aliquot of 100 mg of slurry or 200 µl for the liquid samples was used to isolate DNA using the FavorPrep Stool Kit (Cat.# FASTI001-1; Favorgen Biotech Corp; Ping-Tung, Taiwan), recovering the DNA in 80 µL of elution buffer (ddH2O), which was then stored at −70 °C. DNA concentration was measured using the NanoDrop 2000 spectrophotometer (Cat# ND2000 Thermo Scientific; Massachusetts), and the DNA quality was evaluated by electrophoresis in 0.5% agarose gel. The average yield for the DNA extraction was 37.06 ng/µL.

Semiconductor DNA Sequencing of V3-16S rDNA Libraries
The rDNA libraries amplifying the V3 polymorphic region of the 16S rRNA gene were prepared by PCR using barcoded primers, and semiconductor high-throughput DNA sequencing was performed in the Ion Torrent PGM system as previously described [6].

Data Analysis
The data obtained from the sequencing were analyzed using the QIIME program (Quantitative Insights Into Microbial Ecology, v1.9.0) pipeline [7] to determine the relative abundance of bacteria. The alpha diversity was characterized with the Shannon, Simpson and Chao1 indices, and we observed species using the phyloseq (v1.22.3) and ggplot2 (v3.1.0) packages in the R program (v3.4.4) [8]. The beta diversity dissimilarity index was calculated by the UniFrac distance metric as a % of the total variability in different axes of the plot and visualized by principal coordinate analysis as described in Ref. [9].

Abundance of Phyla in the Ready-to-Eat Food
We found a great bacterial diversity among all studied ready-to-eat dishes ( Table 1). The most abundant bacterial phyla were Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, TM7 and Thermi, among others. In the dairy products (DA), for example, the phyla Firmicutes and Proteobacteria were the most abundant, except for DA1, which corresponds to a sample of Kefir that exhibited a large abundance of the phylum Proteobacteria (Figure 1). A similar situation was observed for the side dishes (SD) where the SD3 corresponding to a sample of "chef's salad" was the one with a large relative abundance of Proteobacteria. The soups such as SO1 and SO5 (Table 1) had a large abundance of Proteobacteria and Bacteroidetes, respectively, while the soups SO2, SO3, and SO4 exhibited similar large abundances of Firmicutes (Figure 1). The main dishes MD1 to MD7 (Table  1), including dishes consisting mostly of the cooked meat of chicken, pork or beef, had Firmicutes as the most abundant phylum, with the exception of MD2 (soft taco with pork rind), which had Proteobacteria as the most abundant phylum. When the microbiota of the sample of desserts DE1 to DE6 were analyzed, again the Firmicutes were the most abundant phyla with the exception of "sugary churros" (DE1), which contained a high abundance of Actinobacteria, and "chocolate breadflan" (DE6) which contained Proteobacteria (Figure 1). The three beverages we studied (Table 1), made mostly with uncooked vegetable ingredients, showed high abundances of Firmicutes (BE1 and BE3) and Proteobacteria (BE2) (Figure 1).

Discussion
In this work we characterized the bacterial diversity present in ready-to-eat Mexican dishes, habitually consumed by adult Mexican workers. We found a remarkable bacterial diversity in the unspoiled food from which DNA was extracted after homogenization, with bacterial members of the phyla Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, TM7, and Thermi. The Mexican healthy adult population who consumes this type of food have a fecal microbiota characterized by members of the phyla Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria, which commonly includes members of the order Rhizobiales, Cytophagales, Nitrospirales, families f_Sphingomonadaceae, f_Cytophagaceae, f_Chitinophagaceae and f_Sphingomonadaceae, and genera such as Bulleidia, Agrobacterium, Lentzea, Nitrospira, and Sphingomonas [9]. We believe that in addition to environmental and genetic factors, every day food supplies a defined set of bacteria wherein some members either get established in the gut or influence the establishment of other taxa.

Conclusions
Based on our results, we conclude that unspoiled ready-to-eat Mexican dishes contain a richly diverse bacterial community, which may contribute to the organoleptic properties of the dishes, and also might contribute to the gut microbiota on a daily basis, without representing a sanitary risk for the consumers.