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

Abstract

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.

1. 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.

2. Materials and Methods

2.1. 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. Ready-to-eat samples.

ID	Local Mexican Spanish Name	English Name	Description
DA1	Leche-Búlgara	Kefir	Fermented dairy product.
DA2	Leche-Liconsa	Milk	Synthetic milk.
DA3	Leche-Saborizada	Flavored milk	Strawberry flavored milk.
DA4	Yogurt-bebible	Drinkable yogurt	Yogurt with a liquid consistency.
DA5	Yogurt-Griego	Greek style yogurt	Yogurt with a higher amount of fat than normal.
SD1	Ensalada-de-pollo	Chicken salad	Vegetable salad that includes chicken for protein.
SD2	Ensalada-de-jamón	Ham salad	Vegetable salad that includes ham as a source of protein.
SD3	Ensalada-del-chef	Chef’s salad	Vegetable salad with chicken, cubed cheese, and boiled egg.
SD4	Ensalada-de-frutas	Salad with fruits	Vegetable salad that includes fruits within its ingredients.
SD5	Ensalada-de-broccoli	Broccoli salad	Steamed broccoli with fresh vegetables.
SO1	Sopa-de-papa	Potato soup	Tomato broth with diced potato chunks.
SO2	Arroz-a-la-Mexicana	Mexican rice	Rice with tomato, carrot, and pea as main ingredients.
SO3	Crema-de-poblano	Poblano cream	Crepe prepared with poblano pepper.
SO4	Sopa-de-verduras	Vegetable soup	Tomato broth with diced vegetables.
SO5	Arroz-chino	Chinese-style rice	Rice prepared as a typical oriental recipe.
MD1	Alambre	Beef kabob cooked on a grill.	Cubes of broiled beef, bell pepper, onion, bacon, and melted cheese.
MD2	Taco-de-canasta con chicharrón	Soft taco with pork rind.	Steamed soft corn tortillas stuffed with fried pork rind.
MD3	Huevo-con-chorizo	Egg with chorizo	Fried egg accompanied by Spanish sausage.
MD4	Taco-de-guisado	Stew taco	Taco with Mexican style-rice with sausages with tomato.
MD5	Torta-de-jamón	Mexican ham torta	Mexican bolillo (crusty roll) filled with mayonnaise, avocado, ham, and basket cheese.
MD6	Enfrijoladas	Mexican enfrijoladas	Fried tortillas, dipped in a slurry of refried beans, and stuffed.
MD7	Torta-de-pierna	Mexican pork leg torta	Mexican bolillo (crusty roll) filed with mayonnaise, beans, avocado, tomato, smoked pork leg.
DE1	Churros-azucarados	Sugary churros	Fried-dough pastry in oil, with granulated sugar.
DE2	Pay-de-Piña	Pineapple Pie	Pie filled with pineapple jam.
DE3	Frituras de maíz con limón y sal	Corn chips with lemon and salt	Deep fried corn flour chips with lemon and salt.
DE4	Dona glaseada	Glassed donut	Dough fried in oil and glazed with sucrose after cooking.
DE5	Gorditas-de-la-Villa	Villas’s soft cookies	Cornmeal (cacahuazintle), sucrose, baking soda, eggs, vanilla essence, water, and vegetable shortening.
DE6	Chocoflan	Chocolate bread-flan	Chocolate flavor bread with a layer of sweetened egg custard with caramel topping.
BE1	Jugo de naranja	Orange juice	Juice obtained from orange fruit.
BE2	Agua de melón	Melon water	Water accompanied by blended melon and sugar to sweeten.
BE3	Jugo verde	Green juice	Juice obtained by blending spinach, pineapple, orange juice and celery.

DA, dairy product; SD, side dishes; SO, soups; MD, main dishes; DE, deserts; BE, beverages.

).

2.2. 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.

2.3. 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].

2.4. 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].

3. Results

3.1. 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 bread-flan” (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).

3.2. Abundance of Orders, Families, and Genera in the Ready-to-Eat Food

Among the most abundant taxa, the families Streptomycetaceae (Actinobacteria) and Rikenellaceae (Bacteroidetes), and the genera Oscillospira (Firmicutes), Prevotella (Bacteroidetes), Faecalibacterium (Firmicutes), Bacteroides (Bacteroidetes), and Pseudomonas (Proteobacteria), were present in almost all the food dish categories (Figure 2). In the DA group, the “Kefir sample” (DA1) showed high abundances of Enterococcus (Firmicutes), Erisipelotrichaceae (Firmicutes), f Leuconostocaceae (Firmicutes), Pseudomonas (Proteobacteria), Lactococcus (Firmicutes), Acinetobacter (Proteobacteria), Lactobacillales (Firmicutes), Acetobacter (Proteobacteria), and f S24_7 (Bacteroidetes); while a “drinkable yogurt” (DA4) had high abundances of Oscillospira (Firmicutes), Streptomycetaceae (Actinobacteria), Prevotella (Bacteroidetes), Faecalibacterium (Firmicutes), Bacteroides (Bacteroidetes), Weisella (Firmicutes), Staphylococcus (Firmicutes), Rikenellaceae (Bacteroidetes), and Aeromonadaceae (Proteobacteria). Two side dishes had remarkable abundances of bacteria; for instance, the “chicken salad” (SD1) had Thermus (Deinococcus–Thermus), Leuconostocaceae (Firmicutes), Acetobacteraceae (Proteobacteria), and Geobacillus (Firmicutes), while the “chef’s salad” (SD3) exhibited high abundances of Staphylococcus (Firmicutes), Rikenellaceae (Bacteroidetes), Lactobacillales (Firmicutes), and Acetobacteraceae (Proteobacteria). In the case of soups, only the “Chinese-style rice” (SO5) had a comparable high abundance of Bacteroides (Bacteroidetes). Main dishes such as “soft taco with pork rind” (MD2) had high abundances of Aeromonadaceae (Proteobacteria), Acetobacteraceae (Proteobacteria), and Geobacillus (Firmicutes); this last, Firmicutes, was also observed in high abundance in “Egg with chorizo” (MD3). The f S24_7 (Bacteroidetes), Acetobacteraceae (Proteobacteria), and g Geobacillus (Firmicutes) were abundant in the “Stew taco dish” (MD4); while in the same category, the “Mexican ham torta”, “Mexican enfrijoladas” and “Mexican pork leg torta” carried high abundances of Weisella (Firmicutes), Thermus (Deinococcus–Thermus), Acetobacter (Proteobacteria), and Acetobacteraceae (Proteobacteria). Among the sweets, only the “Chocolate bread-flan” carried Pseudomonas (Proteobacteria) in a high abundance comparable to the other dishes, and the same was observed in the beverages, where only the orange juice carried a comparably high abundance of Lactococcus (Firmicutes) (Figure 2).

4. 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.

5. 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.