# Jenna M. Lang (jennomics@gmail.com). Genome Center, University of California, Davis, CA, USA Jonathan A. Eisen (jaeisen@ucdavis.edu). Genome Center, Evolution and Ecology, Medical Microbiology and Immunology, University of California, Davis, Davis, CA, USA Angela M. Zivkovic (amzivkovic@ucdavis.edu). Department of Nutrition, University of California, Davis, CA, USA

Abstract

#Abstract

Far more attention has been paid to the microbes in our feces than the microbes in our food. Research efforts dedicated to the microbes that we eat have historically been focused on a fairly narrow range of species, namely those which cause disease and those which are thought to confer some "probiotic" health benefit. Little is known about the effects of ingested microbial communities that are present in typical American diets, and even the basic questions of which microbes, how many of them, and how much they vary from diet to diet and meal to meal, have not been answered. We characterized the microbiota of three different dietary patterns in order to estimate: the average total amount of daily microbes ingested via food and beverages, and their composition in three daily meal plans representing three different dietary patterns. The three dietary patterns analyzed were: 1) the Average American (AMERICAN): focused on convenience foods, 2) USDA recommended (USDA): emphasizing fruits and vegetables, lean meat, dairy, and whole grains, and 3) Vegan (VEGAN): excluding all animal products. Meals were prepared in a home kitchen or purchased at restaurants and blended, followed by microbial analysis including aerobic, anaerobic, yeast and mold plate counts as well as 16S rRNA PCR survey analysis. Based on plate counts, the USDA meal plan had the highest total amount of microbes at $$1.3 X 10^9$$ CFU per day, followed by the VEGAN meal plan and the AMERICAN meal plan at $$6 X 10^6$$and $$1.4 X 10^6$$ CFU per day respectively. There was no significant difference in diversity among the three dietary patterns. Individual meals clustered based on taxonomic composition independent of dietary pattern. For example, meals that were abundant in Lactic Acid Bacteria were from all three dietary patterns. Some taxonomic groups were correlated with the nutritional content of the meals. Predictive metagenome analysis using PICRUSt indicated differences in some functional KEGG categories across the three dietary patterns and for meals clustered based on whether they were raw or cooked. Further studies are needed to determine the impact of ingested microbes on the intestinal microbiota, the extent of variation across foods, meals and diets, and the extent to which dietary microbes may impact human health. The answers to these questions will reveal whether dietary microbial approaches beyond probiotics taken as supplements - i.e., ingested as foods - are important contributors to the composition, inter-individual variation, and function of our gut microbiota.

#Introduction
The human gut microbiome (the total collection of microbes found in in the human gut) mediates many key biological functions and its imbalance, termed dysbiosis, is associated with a number of inflammatory and metabolic diseases from inflammatory bowel disease to asthma to obesity and insulin resistance (Machonkin 2014) (Costello 2012). How to effectively shift the microbiome and restore balance is a key question for disease prevention and treatment. The gut microbiome is influenced by a number of factors including the nature of the initial colonization at birth (e.g., vaginal vs. C-section delivery), host genotype, age, and diet. As diet is a readily modifiable factor, it is an obvious target for interventions. Several studies have confirmed high inter-individual variability in the bacterial composition of the gut microbiome in healthy individuals (Brownawell 2012), (Costello 2009). Despite this high variability at the species level, enterotypes, or distinct clusters at the genus level, were described as core microbiomes that are independent of age, gender, nationality, or BMI (Arumugam 2011). Although the concept of enterotypes is itself controversial, diet has been shown to play a key role in determining enterotype (Wu 2011, Filippo 2010, Muegge 2011). Although the core microbiota within each person are stable over longer time scales (e.g. 5 years), community composition is highly dynamic on shorter time scales (e.g. 0–50 weeks) (Faith 2013). In fact, major shifts occur within 1 day of a significant dietary change (Wu 2011, Turnbaugh 2009). “Blooms” in specific bacterial groups were observed in response to controlled feeding of different fermentable fibers (Walker 2011). Dietary changes affect both the structure and function of the gut microbiome in animals (Hildebrandt 2009), and humans under controlled feeding conditions (Wu 2011). Rapid shifts in microbiome composition are observed in response to change from a vegetarian to an animal based diet (David 2013).

An ecological perspective helps to delineate the complexity and multi-layered nature of the relationships between the microbiota, the human host, and both the nutritive and non-nutritive compounds we ingest (Costello 2012). The concept of the human gut microbiome as a distinct ecosystem or collection of micro-ecosystems allows us to identify and characterize the components of the system, including its inputs and outputs. In this case, the inputs of the system include all of the various ingested compounds that can either serve as food substrates (e.g. complex sugars) or that can be metabolized by or that affect the metabolism of the microbiota (e.g. polyphenolic compounds, environmental chemicals, medications). Some of these inputs, such as probiotics have been studied extensively. It has been well documented that certain sugars such as galactooligosaccharides, fructooligosaccharides, and oligosaccharides found in milk act as prebiotics that support the establishment and growth of certain commensal microbial species (Brownawell 2012), (de 2008), (Roberfroid 2007), (German 2008), (Zivkovic 2011). Research has also documented the effects of antibiotics, and pathogens on the microbiota composition, its recovery or lack of recovery to baseline following resolution, and the various immunological and physiological effects of these perturbations (Manichanh 2010), (Ubeda 2012), (Bien 2013)(Dethlefsen 2010).

Yet, little is known about the effects of ingested microorganisms on gut microbiota composition or function, and even the basic questions of which microbes, how many of them, and how much they vary from diet to diet and meal to meal, have not been answered. We do know about the microbial ecology of various specialty foods where fermentation, colonization, ripening, and/or aging are part of the preparation of these foods, for example pancetta (Busconi 2014) and of course cheese (Gatti 2008)(Button 2012). The microbial ecology of the surfaces of raw plant-derived foods such as fruits and vegetables has also been characterized (Leff 2013). There is a large base of literature on food-borne pathogens (Aboutaleb 2014). Furthermore, it is known that the microbial ecology of endemic microbes found on food surfaces can affect mechanisms by which pathogens colonize these foods (Critzer 2010). A recent article showed that certain ingested microbes found in foods such as cheese and deli meats were detected in the stool of individuals who consumed them, and that furthermore they were culturable and thus survived transit through the upper intestinal tract (David 2013). However, the microbial ecology or microbial assemblages of different meals and diets, as well as the total number of live microorganisms ingested in these meals and diets are largely unknown. In fact, studies of the effects of diets and foods on the gut microbiota rely on dietary recalls and other dietary reporting instruments that were not designed to capture the potential variability in aspects of foods other than their basic macronutrient and micronutrient content. Specifically, current instruments for collecting individual dietary data do not capture the provenance of foods or their preparation, both of which would likely influence certain compositional aspects of the foods, especially the microbes on those foods.

We performed a preliminary study designed to generate hypotheses about the microbes we eat, and how they vary in terms of total abundance and relative composition in different meals and dietary patterns typical of American dietary intakes. We have selected to characterize the microbiota of 15 meals that exemplify the typical meals consumed as part of three different dietary patterns in order to determine the average total amount of daily microbes ingested via food and beverages and their composition in the average American adult consuming these typical foods/diets: 1) the Average American dietary pattern (AMERICAN) focused on convenience foods, 2) the USDA recommended dietary pattern (USDA) emphasizing fruits and vegetables, lean meat, dairy, and whole grains, and 3) the Vegan (VEGAN) dietary pattern, which excludes all animal products. We used DNA sequencing, plate counting, and informatics methods to characterize microbes in these meals and dietary patterns.

#Methods
##Meal preparation
We conducted a series of experiments consisting of food preparation followed by sample preparation and microbial analysis. Food was purchased and prepared in a standard American home kitchen by the same individual using typical kitchen cleaning practices including hand washing with non-antibacterial soap between food preparation steps, washing of dishes and cooking instruments with non-antibacterial dish washing detergent, and kitchen clean-up with a combination of anti-bacterial and non-antibacterial cleaning products. Anti-bacterial products had specific anti-bacterial molecules added to them whereas "non-antibacterial" products were simple surfactant-based formulations. The goal was to simulate a typical home kitchen rather than to artificially introduce sterile practices that would be atypical of how the average American prepares their meals at home. All meals were prepared according to specific recipes (from raw ingredient preparation such as washing and chopping, to cooking and mixing).

After food preparation, meals were plated on a clean plate, weighed on a digital scale (model 157W, Escali, Minneapolis, MN), and then transferred to a blender (model 5200, Vita-Mix Corporation, Cleveland, OH) and processed until completely blended (approximately 1-3 minutes). Prepared, ready to eat foods that were purchased outside the home were simply weighed in their original packaging and then transferred to the blender. 4 mL aliquots of the blended meal composite were extracted from the blender, transported on dry ice and then stored at -80°C until analysis. The following analyses were completed using these meal composite samples: 1) total aerobic bacterial plate counts, 2) total anaerobic bacterial plate counts, 3) yeast plate counts,
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