Location: Dairy and Functional Foods Research
2020 Annual Report
Objectives
1: Establish a gut microbiota community utilizing the twin-Simulator of the Human Intestinal Microbial Ecology (TWINSHIME®) system.
2: Evaluate changes in the bacterial a) community composition b) metabolome, and c) proteome in response to dietary components.
2a: Evaluate changes in the population composition, metabolome, and proteome of the gut microbiota using the TWINSHIME® in response to fluctuations in the pH of the large intestinal regions.
2b: Evaluate alterations in the population composition, metabolome, and proteome of the gut microbiota using the TWINSHIME® in response to defined dietary interventions. In particular fat-free or full-fat milk and whole wheat or refined wheat flour.
Approach
This project will study the effects of dietary components on the human gut microbiota of the large intestine. This will be done using the TWINSHIME® system, which is a dynamic, in vitro system capable of simulating the physiological conditions of the human gastrointestinal tract. This system is comprised of two sets of bioreactors (SHIME 1 and 2) arranged in parallel to mimic the stomach, small intestine, and the large intestine, which is divided into the ascending, transverse, and descending regions. In the first objective, the ability for the TWINSHIME® system to establish a stable human gut microbiota community, representative of the distinct regions of the large intestine will be examined. Data from Next Generation DNA sequencing and short chain fatty acids (SCFA) analysis will be used to confirm stability and demonstrate that once it is achieved, it remains the same until the experiment is terminated. Furthermore, the proteomics and metabolomics research will enable establishment of the correlation of the change in composition with specifically functional expression of gut microbiota. In the second objective, changes in the bacterial a) community composition b) metabolome, and c) proteome, in response to alterations of pH and the dietary components milk and whole wheat will be measured. To analyze the response of the microbiota to changes in pH, the pH of the colon reactors in SHIME 1 will be lowered, while the pH of the colon reactors in SHIME 2 will be increased. In order to determine changes to the bacterial community, metabolome, and proteome in response to milk, SHIME 1 will be supplemented with 8 ounces of fat-free milk per feeding and SHIME 2 supplemented with 8 ounces of full-fat milk per feeding. In order to test the effect of wheat flour on the gut microbiota population and/or metabolome, SHIME 1 will be supplemented with refined wheat flour and SHIME 2 will be supplemented with whole wheat flour. During these experiments, samples will be harvested every three days and subjected to analysis. Data from 16S rRNA sequencing will be used to determine community composition; Gas and Liquid chromatography, coupled with mass spectrometry, and a MALDI-TOF/TOF-MS/MS will be used to determine changes in the metabolome (SCFA, amino acids, volatiles, peptides, sugars, and lipids) and the proteome. By compiling these results, the effects of pH change, milk, and wheat on the community composition, metabolome and proteome of the gut microbiota of the individual colon regions can be accurately determined.
Progress Report
This is the final report for Project 8072-41000-102-00D, which will end on June 30, 2020. The new NP306 project entitled “In vitro Human Gut System: Interactions Between Diet, Food Processing, and Microbiota” has been certified by OSQR and is currently being established.
A stable human microbial community of the large intestine was established in vitro using the Twin Simulator of Human Intestinal Microbial Ecosystem (TWINSHIME®) inoculated with a fecal sample from a healthy donor. The composition of the gut microbial communities in the ascending, transverse, and descending colon regions, for both the luminal and mucosal phases, were determined using 16S rRNA gene sequencing, and the levels of short-chain fatty acids were quantified by GC/MS. Based on these results, it was concluded that the established, in vitro gut microbial community reaches a steady-state approximately two weeks after inoculation and that this steady-state can be maintained for at least an additional five weeks. This experiment was repeated 4 times, and the results were consistent. Three research papers were published. The results of this experiment fulfill the requirements of the milestones for Objective 1.
Research on the composition and metabolic changes of the gut microbiota in response to fluctuations in the pH of the large intestinal regions focused on Objective 2, bench experiments, analysis of microbial composition and metabolites, as well as the bioinformatics analysis of DNA sequencing, was completed. The results of these experiments led to the conclusion that environmental pH has a significant impact on the structure of the gut microbial community by altering diversity and influencing levels of taxonomic abundance. In addition, changes to the environmental pH resulted in significant alteration of both the quantity and type of short chain fatty acids produced. The drafts of two manuscripts that summarize the experimental results are in preparation.
Several short-term experiments were performed simultaneously with the larger experiments to fill the void between larger experiments, which served to enrich the findings for each objective and promoted productivity. These short-term experiments included research on the response of the gut microbial community to stevia, triclosan, and the metagenomic assessment of the Cebus apella gut microbiota. Additionally, we have been working on a model of the small intestinal gut microbiota in collaboration with a group from U Penn. The results are reported in the following paragraphs.
Leaf extracts of Stevia rebaudiana (steviol glycosides, SGs) are used as non-nutritive, table sugar (sucrose) alternatives due to their high level of sweetness and low caloric impact. Little is known of the impact of the SGs on the human gut microbiota in terms of diversity, composition, and metabolic products. There have been recent questions about safety in terms of overall health with respect to the use of non-nutritive sweeteners in general and SGs in particular. Testing of SGs and erythritol using six representatives of the gut microbiota in vitro found no impact on bacterial growth, yet treatment with erythritol resulted in an enhancement of butyric and pentanoic acid production when tested using a human gut microbial community. Furthermore, the administration of SGs and erythritol to a Cebus apella model resulted in changes to the gut microbial structure and diversity. Overall, the study did not find a negative impact of SGs and erythritol on the gut microbial community. This study is the first to combine in vitro and in vivo work on the impact of SGs in commercial products on the gut microbiota. This is also the first study to show that SGs can have an observable impact on the alpha and beta diversity of the gut microbial community in vivo. One paper was published based on these findings.
The recent ban of the antimicrobial compound Triclosan (TCS) from use in antimicrobial consumer soaps followed research that showed the risk it poses to the environment and human health. TCS has been found in human plasma, urine, and milk, demonstrating that it is present in human tissues. Previous work has also demonstrated that the consumption of triclosan disrupts the gut microbial community of mice and zebrafish. Due to the widespread use of TCS and ubiquity in the environment, it is imperative to understand the impact this chemical has on the human body and its symbiotic resident microbes. To that end, this study is the first to explore how Triclosan impacts the human gut microbial community in vitro both during and after treatment. Through our in vitro system simulating three regions of the human gut; the ascending colon, transverse colon, and descending colon regions, we found that treatment with triclosan significantly impacted the community structure in terms of reduced population, diversity, and metabolite production, most notably in the ascending colon region. Given a 2-week period of stopping TCS use, most of the population levels, community structure, and diversity levels were recovered for all colon regions. Our results demonstrate that the human gut microbial community diversity and population size is significantly impacted by TCS at a high dose in vitro and that the community is recoverable within this system. One paper was published based on this research.
Intestinal metabolic profiles are specifically correlated with regionally developed community composition and genetic potential of the gut microbiome. The colon gut microbiota is responsible for complex chemical conversions of nutrients and subsequent release of metabolites. Here, gas and liquid chromatography coupled with mass spectrometry was applied to generate the metabolic profiles of the region-specific microbial communities cultured using an in vitro platform simulating the ascending (AC), transverse (TC), and descending (DC) colon regions. Comparative analysis revealed a large divergence between the metabolic profiles of the AC region to the TC and DC regions in terms of short-chain fatty acid production, metabolic spectrum, and conversion of primary to secondary bile acids. Metagenomic evaluation revealed that the regionally derived metabolic profiles had a strong correlation to community composition and genetic potential. Together, the results provide key insights regarding the metabolic divergence of the regional communities that are integral to understand the structure-function relationship of the gut microbiota. The methodology developed in the research and the results thus obtained are of significance to food and nutrition sciences, pharmaceutical science and new drug development.
Cebus Apella (C. apella) is a species of Nonhuman Primate (NHP) used for biomedical research because it is phylogenetically similar and shares anatomical commonalities with humans. The gut microbiota of three C. apella were examined in the different regions of the intestinal tract. Using metagenomics, the gut microbiota associated with the luminal content and mucus layer for each intestinal region was identified, and functionality was investigated by quantifying the levels of short-chain fatty acids (SCFAs) produced. The results of this study show a high degree of similarity in the intestinal communities among C. apella subjects, with multiple shared characteristics. First, the communities in the lumen were more phylogenetically diverse and rich compared to the mucus layer communities throughout the entire intestinal tract. The small intestine communities in the lumen displayed a higher Shannon diversity index compared to the colon communities. Second, all the communities were dominated by aero-tolerant taxa such as Streptococcus, Enterococcus, Abiotrophia, and Lactobacillus, although there was preferential colonization of specific taxa observed. Finally, the primary SCFA produced throughout the intestinal tract was acetic acid, with some propionic acid and butyric acid detected in the colon regions. The small intestine microbiota produced significantly less SCFAs compared to the communities in the colon. Collectively, these data provide an in-depth report on the composition, distribution, and SCFA production of the gut microbiota along the intestinal tract of the C. apella NHP animal model. One paper was published based on the research.
There has been continuing development on an in vitro small intestinal model, in collaboration with researchers at the University of Pennsylvania. In 2018, for the first time, the gut microbiota of the small intestine and the large intestine were cultured in vitro and run in parallel. The kinetics of the bacterial communities were compared, in terms of population dynamics and functional properties, genetic potential elucidated, and the effect of physiological levels of oxygen was analyzed. This research has been expanded to study the differential RNA expression patterns of the small intestine gut microbial community in anaerobic vs. aerobic conditions. This has required the development of novel methods to analyze changes in RNA expression patterns between complex communities and has provided data on how facultative taxa within the small intestine gut microbial community responds to oxygen. One research paper is in preparation.
Accomplishments
1. Dysbiosis caused by Triclosan (TCS) can be reversed simply by stopping the use of the chemical. Triclosan is an anti-bacterial chemical compound whose wide usage over the decades in consumer products, such as soap and toothpaste, and its ability to be absorbed by the human body has led to concerns about its impact on human health. To address these concerns, ARS scientists at Wyndmoor, Pennsylvania, studied the effect of triclosan on the bacterial community of the human colon. To do so, the scientists used an in vitro culture system named the “Twin Simulator of the Human Intestinal Microbial Ecosystem (TWINSHIME®)” to evaluate whether prolonged exposure of human gut microbiota to TCS would have a large effect on the bacterial community, as well as whether that effect could be reversed after ending treatment. It was found that the introduction of TCS into the bacterial community caused a sharp decline in bacterial population and composition, as well as the compounds they produce. Most importantly, the scientists showed for the first time that the decrease in bacterial population, composition, and compounds was reversible over a 2-week period of stopping TCS use with a rebalancing of the gut microbial community.
2. Stevia has no negative effect on the gut. Stevia is a popular plant-based low-calorie sweetener loved by consumers on low carbohydrate diets. Stevia is a natural sweetener because the compounds that deliver sweetness are extracted from the leaves of the Stevia plant using water or ethanol and then dried. There have been recent questions about the safety in terms of overall health with respect to use of non-nutritive sweeteners such as Stevia exracts. ARS scientist at Wyndmoor, Pennsylvania, studied the effect of stevia on models of human gut bacteria and found that stevia has no adverse impact on the bacteria in the human gut models.
Review Publications
Muhidinov, Z.K., Bobokalonov, J.T., Ismoilov, I.B., Strahan, G.D., Chau, H.K., Hotchkiss, A.T., Liu, L.S. 2020. Characterization of two types of polysaccharides from Eremurus hissaricus roots growing in Tajikistan. Food Hydrocolloids. 205. https://doi.org/10.1016/j.foodhyd.2020.105768.
Firrman, J., Tanes, C., Bittinger, K., Mahalak, K.K., Rinaldi, W., Liu, L.S. 2019. Metagenomic assessment of the Cebus Apella gut microbiota. American Journal of Primatology. https://doi.org/10.1002/ajp.23023.
Mahalak, K.K., Firrman, J., Lee, J., Bittinger, K., Nunez, A., Bobokalonov, J., Arango-Argoty, G., Zhang, L., Zhang, G., Liu, L.S. 2020. Triclosan has a robust, yet reversible impact on human gut microbial composition in vitro. PLoS One. 15(6): 1-22. https://doi.org/10.1371/journal.pone.0234046.
Firrman, J., Liu, L.S., Tanes, C., Friedman, E., Bittinger, K., Daniel, S., Van Den Abbeele, P., Evans, B. 2019. Metabolic analysis of the regionally distinct gut microbial communities using an in vitro platform. Journal of Agriculture and Food Sciences. Pages A-L. https://doi.org/10.1021/acs.jafc.9b05202.
Mahalak, K.K., Firrman, J., Tomasula, M.M., Nunez, A., Lee, J., Bittinger, K., Rinaldi, W., Liu, L.S. 2019. Impact of steviol glycosides and erythritol on the human and Cebus apella gut microbiome. Journal of Agricultural and Food Chemistry. Pages A-I. https://doi.org/10.1021/acs.jafc.9b06181.
Zhou, S., Jin, Z.T., Sheen, S., Zhao, G., Liu, L.S., Juneja, V.K., Yam, K. 2020. Development of sodium chlorite and glucono delta-lactone incorporated PLA film for microbial inactivaton on fresh tomato. Food Research International. 132:1-7. https://doi.org/10.1016/j.foodres.2020.109067.
