Large Quantities of Bacterial DNA and Protein in Common Dietary Protein Source Used in Microbiome Studies

Diet has been shown to greatly impact the intestinal microbiota. To understand the role of individual dietary components, defined diets with purified components are frequently used in diet-microbiota studies. Many of the frequently used defined diets use purified casein as the protein source. Previous work indicated that this casein contains microbial DNA potentially impacting results of microbiome studies. Other diet-based microbially derived molecules that may impact microbiome measurements, such as proteins detected by metaproteomics, have not been determined for casein. Additionally, other protein sources used in microbiome studies have not been characterized for their microbial content. We used metagenomics and metaproteomics to identify and quantify microbial DNA and protein in a casein-based defined diet to better understand potential impacts on metagenomic and metaproteomic microbiome studies. We further tested six additional defined diets with purified protein sources with an integrated metagenomic-metaproteomic approach and show that contaminating microbial protein is unique to casein within the tested set as microbial protein was not identified in diets with other protein sources. We also illustrate the contribution of diet-derived microbial protein in diet-microbiota studies by metaproteomic analysis of stool samples from germ-free mice (GF) and mice with a conventional microbiota (CV) following consumption of diets with casein and non-casein protein. This study highlights a potentially confounding factor in diet-microbiota studies that must be considered through evaluation of the diet itself within a given study. ImportanceMany diets used in diet-microbiota studies use casein as the source of dietary protein. We found large quantities of microbial DNA and protein in casein-based diets. This microbial DNA and protein are resilient to digestion as it is present in fecal samples of mice consuming casein-based diets. This contribution of diet-derived microbial DNA and protein to microbiota measurements may influence results and conclusions and must therefore be considered in diet-microbiota studies. We tested additional dietary protein sources and did not detect microbial DNA or protein. Our findings highlight the necessity of evaluating diet samples in diet-microbiota studies to ensure that potential microbial content of the diet can be accounted for in microbiome measurements.