Location: Dairy and Functional Foods Research
2018 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
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 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. The results of this experiment fulfill the requirements of the milestone for Objective 1.
To further verify the applicability of the TWINSHIME to study the region-specific and phase-specific gut microbial communities, an in vitro and in vivo parallel study was conducted using an animal model. Briefly, fecal samples were collected and used to cultivate an in vitro animal intestinal microbial community in the TWINSHIME system. The composition of the microbial communities that developed for both the luminal and mucosal phases in the TWINSHIME bioreactors representing the ascending, transverse, and descending colon regions were compared with the corresponding communities obtained from the animals. This set of experiments is ongoing. The results of this experiment will exceed the requirements of the milestone for Objective 1.
In an external collaboration for this project, the gut microbiota community of the small intestine was cultured in vitro using a single chemostatic bioreactor inoculated with an ileostomy sample harvested from an adult human. Using 16S rRNA gene sequencing, it was determined that, similar to the large intestine, this community was able to reach a steady state within two weeks of inoculation. The production of short chain fatty acids, consumption of amino acids, and bile acid utilization was determined by LC/MS. The results of this experiment contribute to the milestone for Objective 1.
In order to evaluate the effect of pH changes on the region and phase specific communities of the gut microbiota, a stable human gut microbial community was cultured using the TWINSHIME system. After stabilization, the pH for each intestinal region for the first SHIME unit was decreased by 0.5 pH, and each intestinal region of the second SHIME unit was increased by 0.5 pH. After two weeks, the pH for each intestinal region of the first SHIME unit was decreased by another 0.5 pH, and the pH for each intestinal region of the second SHIME unit was increased by another 0.5 pH. After two weeks, the pH for each intestinal region was reset to the original pH values. The changes to the gut microbial community in response to the increase and decrease in pH was determined by 16S rRNA gene sequencing, and the levels of short chain fatty acids were quantified by GC/MS. This experiment is ongoing, but represents substantial progress towards completion of the milestone for Objective 2.
Using the TWINSHIME, the effects of Splenda (a sugar alternative popularly used in food and beverage products) and Triclosan (an antibacterial chemical used in detergents, cosmetics and furniture, etc.) on the composition of gut microbiota and their metabolome were determined. These experiments are ongoing. The results of these experiments contribute towards objective 2.
The breakdown of the plant polyphenol apigenin by the gut microbiota of the colon was analyzed in vitro using a pair of chemostatic bioreactors. In short, two bioreactors were inoculated with feces from a healthy human donor and cultured for two weeks until stable. Apigenin was then added to one of the bioreactors, and to the other bioreactor served as a control. Samples were harvested over the course of 24 hours, and LC/MS was used to determine the breakdown of apigenin and the resulting byproducts. These experiments are ongoing, and the results contribute to the milestone for Objective 2.
Accomplishments
1. Establishing a stable human large intestine microbiota in vitro. The application of in vitro technology to study the gut microbiota of the large intestine has provided valuable information on the interaction between the members of this community and nutrition. Yet, there is a lack of knowledge regarding the ability of an in vitro culture system to produce a gut microbial community that can not only maintain stability, but is also able to develop region and phase specificity. ARS researchers at Wyndmoor, Pennsylvania established a gut microbial community in a TWINSHIME system and analyzed the community in all three colon regions over the course of a 7-week period. The results of this study demonstrated that the community and its production of short chain fatty acids reached stability within 2 weeks of inoculation and remained stable. For the first time, it was demonstrated that the microbial community that developed in the mucosal phase not only achieved stability, but was also region specific. The in vitro system that was developed can be used to evaluate the effect of diet on the gut microbiota, and how this may contribute to health and the development of disease.
2. Establishment of a human small intestine microbiota in vitro and its response to oxygen. Although it is well known that the gut microbiota of the colon is a mediator between nutrition and human health, there is little known about the gut microbiota that resides in the small intestine and its functions. For the first time, ARS researchers at Wyndmoor, Pennsylvania, in conjunction with researchers at the University of Pennsylvania, have cultured the small intestine gut microbiota in vitro using a chemostatic bioreactor. The small intestine community was compared to a large intestine community, and both were studied without oxygen and with a supply of 5% oxygen. The results demonstrated that the small intestine and colon gut microbial communities were distinct from each other. The addition of oxygen to the small intestine community made its composition more similar to the inoculum, but the addition of oxygen to the large intestine community produced minimal changes. Functionally, the large intestine community had significantly higher short chain fatty acid production and conversion of primary to secondary bile salts compared to the small intestine community.
Review Publications
Li, R., Jin, Z.T., Liu, Z., Liu, L.S. 2018. Antimicrobial double-layer coating prepared from pure or doped-titanium dioxide and binders. Coatings. 8(1):41-51.
Zhou, S., Hu, C., Zhao, G., Jin, Z.T., Sheen, S., Liu, L.S., Yam, K.L. 2018. Novel generation systems of gaseous chlorine dioxide for salmonella inactivation on fresh tomato. Food Control. 92:479-487. https://doi.org/10.1016/j.foodcont.2018.05.025.
Cai, C., Liu, L.S., Fu, Y. 2017. Processable conductive and mechanically reinforced polylactide/graphene bionanocomposites through interfacial compatibilizer. Polymer Composites. https://doi.org/10.1002/pc.24663.
Liu, L.S., Firrman, J., Tanes, C., Bittinger, K., Thomas-Gahring, A.E., Wu, G.D., Van Den Abbeel, P., Tomasula, P.M. 2018. Establishing a mucosal gut microbial community in vitro using an an artificial simulator. PLoS One. 13(7):1-20. https://doi.org/10.1371/journal.pone.0197692.
