Location: Dairy and Functional Foods Research
2019 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
The research on the composition and metabolite changes of gut microbiota in response to fluctuations in the pH of the large intestinal regions was started in August 2018, suspended in December of the year, due to government shutdown, restarted in February 2019, and now is in progress. The experiments will be completed by August 23, 2019.
The research on the composition and metabolite changes of gut microbiota in response to various types of processed milks was started in March 2019 and is in progress. This research has gained attention from stakeholders and resulted in the development of a collaboration.
There has been continuing development on the in vitro small intestinal model, in collaboration with researchers at University of Pennsylvania. Last year, 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. In the past year, 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.
Natural sweetener Stevia and antimicrobial compound Triclosan were tested for their separate effects on the homeostasis of the gut microbiota in vitro in stable gut microbial communities (SGMC) established in the TWINSHIME and/or in a new culture system comprised of Eppendorf Bioreactors (BioFlow 320). The results from this research confirmed the in vivo findings from other research groups that the introduction of both compounds to the SGMC changed composition and functionality. In addition, this research found, for the first time, that these changes could be recovered after discontinuation of the chemicals for two weeks or longer. Since these two compounds have demonstrated both positive and negative effects on health, and their safe use is a hot topic of public debate, the present research provide useful information for policymakers and consumers to determine the appropriate application of these two compounds. Two research papers are in preparation.
Utilization of the TWINSHIME system is key to research that describes the effects of food components in the different regions of the gastrointestinal tract (GIT). However, whether or not the TWINSHIME system produces communities that are representative of the regionally distinct GIT communities is unknown. In order to fully evaluate the TWINSHIME system, we have implemented parallel studies to compare the composition of the gut microbial community along the GIT established using the SHIME system inoculated with the feces of the Monkey to the microbes obtained from the monkey corpses right after euthanasia. This experiment has been carried out twice in different conditions, the research results showed how the colonic milieu influences the functionality and kinetics of the gut microbiome, revealing the limitation of the TWINSHIME apparatus and drawing a boundary for its appropriate use in research. The third experiment is in preparation, and should be concluded by the end of 2020.
Accomplishments
1. Longitudinal metagenomic assessment of the Cebus Apella gut microbiota. The Cebus Apella (C. Apella) is a species of Non-Human Primate (NHP) used for biomedical research because they are phylogenetically similar and share anatomical commonalities with humans. For humans, the gut microbiota is known to be associated with both health and disease; yet little was known regarding the gut microbiota of the C. Apella. ARS scientists in Wyndmoor, Pennsylvania, found (1) The gut microbiota community of the C. apella was dominated by aero-tolerable taxa such as Streptococcus, Enterococcus, Abiotrophia, and Lactobacillus; (2) The communities that developed within the luminal content and mucus layer of the C. Apella intestinal tract were divergent from each other, with the lumen having more phylogenetic diversity and richness than the mucus communities; and (3) The C. apella colon communities produced significantly more short chain fatty acids (SCFAs) than the small intestine communities. The primary SCFA produced was acetic acid, with small levels of propionic acid and butyric acid detected in the colon regions. The research provides fundamental knowledge for the better use of C. Apella in human heath/diseases research.
Review Publications
Firrman, J., Liu, L.S., Arango Argoty, G., Zhang, L., Tomasula, P.M., Wang, M., Pontious, S., Kobori, M., Xiao, W. 2018. Analysis of temporal changes in growth and gene expression for commensal gut microbes in response to the polyphenol naringenin. Microbial Insights. 11:1-12. https://doi.org/10.1177/1178636118775100.
Wang, M., Firrman, J., Zhang, L., Arango-Argoty, G., Tomasula, P.M., Liu, L.S., Xiao, W., Yam, K. 2017. Apigenin impacts growth of the gut microbiota and alters gene expression of Enterococcus. Molecules. 22(8):1292.
Wang, Q., Wu, Z., Zhang, J., Firrman, J., Wei, H., Zhuang, Z., Liu, L.S., Miao, L., Hu, Y., Diao, Y., Xiao, W. 2017. A robust system for production of superabundant VP1 recombinant AAV vectors. Molecular Therapy. 7:146-156. https://doi.org/10.1016/j.omtm.2017.11.002.
Yao, Y., Wu, J., Zhou, H., Firrman, J., Xiao, W., Sun, Z., Li, D. 2018. A deficiency in cathelicidin reduces lung tumor growth in NNK/NTHi-induced A/J mice. American Journal of Cancer Research. 8(7):1190-1199.
Guo, P., Zhang, J., Huang, J., Chew, H., Firrman, J., Sang, N., Diao, Y., Xiao, W. 2018. Rapid AAV neutralizing antibody determination with a cell-binding assay. Molecular Therapy. 13:40-46. https://doi.org/10.1016/j.omtm.2018.11.007.
Firrman, J., Liu, L.S., Van Den Abbeele, P., Tanes, C., Bittinger, K., Tomasula, P.M. 2019. Applying in vitro culturing technology to establish and evaluate the human gut microbiota. Journal of Visualized Experiments. 144:1-12. https://doi.org/10.3791/59054.
Zhou, S., Hu, C., Liu, L.S., Sheen, S., Zhao, G., Yam, K.L. 2018. A novel gaseous chlorine dioxide generating method utilizing carbon dioxide and moisture respired from tomato for Salmonella inactivation. Food Control. 89:54-61. https://doi.org/10.1016/j.foodcont.2018.01.009.
Bobokalonov, J., Liu, Y., Shahrin, T., Liu, L.S. 2018. Transcriptomics analysis on the regulation of tomato ripening by the ethylene inhibitor 1-methylcyclopropene. Journal of Plant Studies. 7(2):49-60. https://doi.org/10.5539/jps.v7n2p49.
