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ARS Home » Northeast Area » Wyndmoor, Pennsylvania » Eastern Regional Research Center » Dairy and Functional Foods Research » Research » Research Project #439225

Research Project: In vitro Human Gut System: Interactions Between Diet, Food Processing, and Microbiota

Location: Dairy and Functional Foods Research

2021 Annual Report

Objective 1: Determine the effects of dietary bovine milk, with and without lactose, on the gut microbiota. Determine changes to the gut microbiota in response to bovine milk, with and without lactose, in terms of population dynamics and metabolome shifts on the A) small intestine microbiota B) colon microbiota. C) Analyze changes to the microbial metabolomes of the small intestine and colon in response to bovine milk, with and without lactose, which may affect human cells by altering cellular morphology or signaling pathways, and evaluate the health impact of these changes through the detection of health associated biomarkers. Objective 2: Explicate the effect of food processing on the gut microbiota. Examine the inter-effects of cheese and the gut microbiota of the A) small intestine and B) colon by assessing changes to the community population dynamics and functionality and evaluating probiotic potential of the cheese bacterial components to colonize the mucosal surface. C) Investigate the effects of polyphenol and fiber combinations alone, and in the form of a food supplemental bar, on the gut microbial colon community composition and functionality.

This project focuses on the effects of diet and food processing on the dynamics of the gut microbial community (both small and large intestines) and metabolome, and consequently, the impact on health or disease. For small intestine fermentation, experiments will be conducted using a set of 5 bioreactors with 1 designated to simulate gastric digestion, followed by duodenal and jejunal digestion, and the other 4 for ileal gut microbiota growth. For colon fermentation, experiments will use the TWINSHIME apparatus, which simulates the physiological conditions of the colon. Inoculum obtained from ileostomy fluid and from fecal samples will be used for inoculation of the small and large intestines, respectively. Specimens will be taken from each bioreactor at designated time points, and separated into bacterial pellets (BP) and supernatant phases (SP). DNA will be extracted from the BP and quantified. The community composition will be determined using Next Generation 16 Small Ribosomal RNA sequencing of the V1V2 region. Shotgun sequencing may be applied to assess genetic capacity of the microbiota and this information may be used to relate community structure to the observed metabolic function. Reads will be clustered at 97% sequence identity to form Operational Taxonomic Units (OTUs). Communities will be compared globally using weighted and unweighted principal coordinating analysis (PCoA) based on the Jaccard index and Bray-Curtis distance, and alpha diversity metrics. Statistical analysis will be carried out in the R language and corrected for false discovery. The SP will be used for measuring metabolites and examining community functionalities at the molecular level. Gas-chromatography, liquid-chromatography, and mass spectrometry will be used for metabolomics, proteomics, and lipidomics research. UPLC-MS/MS will be used for the analysis of amino acid profiles and bile salt conversion and GC-MS will be used for SCFA analysis. Proteins and peptides may also be analyzed using a nano-LC connected to a Q-TOF MS using the ProteinLynx Global Server for a database search. The selection of statistical analysis and data interpretation, such as student t-test, ANOVA, PCA and/or PCoA, depends on the analytical technique, the nature of the data, and the purpose of the specific research. To evaluate the health impact of the intestinal microbial metabolomes, the changes in cell structure, cellular morphology, signaling pathways, and health associated biomarkers will be examined, using cell lines HT-29, CACO-2, LS-174 T, and HInEpC with multiple dilutions of SP. Changes to cell structure will be determined by analyzing intestinal barrier function through measuring cell viability, quantifying transepithelial electrical resistance, examining cell permeability, and the status of tight junction proteins. Changes to the signaling pathway of cells will be determined by comparing the production of pro-inflammatory cytokines, Interleukin (IL)-1alpha, IL-6, IL-8, IL-18, TNF-alpha, and anti-inflammatory cytokines, IL-4, IL-10, and transforming growth factor (TGF)- beta1, TGF-beta2, TGF-beta3,as well as the expression of the MUC-2 and MUC-5AC genes.

Progress Report
Establishing a small intestine model. In collaboration with researchers at the University of Pennsylvania, the Wyndmoor scientists developed an in vitro cultivar model of a stable human small intestinal and colonic microbiota to characterize the establishment of divergent communities using an ileostomy and a fecal sample as inoculums representative of the human small intestine and colon, respectively. The stable communities, with distinctive compositions similar to their inoculums, were established using the same culture conditions. Regional differences in function, such as short-chain fatty acid production and bile acid transformation, were preserved in vitro and were consistent with the greater representation of glycoside hydrolases and bile inducible operons in the colonic community relative to the small intestinal community. As a physiologically relevant environmental stressor, the results show that the continuous infusion of 5% oxygen into the small intestinal community led to a significant increase in Klebsiella pneumonia. Transcriptomics of the small intestinal community revealed an unanticipated finding that oxygen down down-regulated multiple metabolic pathways, including amino acid biosynthesis, ABC transporters, and oxidative phosphorylation. Together with a reduction in cellular proliferation assessed by peak-to-trough ratio, specifically in Klebsiella pneumonia, this gene expression pattern may be due to the complexities in the response of anutrient-deprived community having reached stationary phase growth. In conclusion, the results show that the composition of divergent gut communities in structure and function primarily depends upon the inoculum's composition. These divergences likely exist predominantly in a nutrient-limited state where oxidative metabolic responses lead to complex patterns of gene expression that differ substantially from studies that characterize acute responses in single bacterial species. The results were summarized in a research paper submitted for review. Capsaicin can modulate the human gut microbiota. Previous studies on capsaicin, the bioactive compound in chili peppers, have shown that it may have a beneficial effect in vivo when part of a regular diet. These positive health benefits, including anti-inflammatory potential and protective effects against obesity, are often attributed to the gut microbial community response to capsaicin. However, there is no consensus on the mechanism behind the protective effect of capsaicin. In this study, the Wyndmoor scientists used an in vitro model of the human gut microbiota to determine how regular consumption of capsaicin impacts the gut microbiota. Using a combination of NextGen sequencing and metabolomics, we found that regular capsaicin treatment changed the structure of the gut microbial community by increasing diversity and certain SCFA abundances, particularly butanoic acid. These changes are responsible for the health benefits associated with CAP consumption. The results of this research were summarized in a research paper submitted for review. Persistence of LGG in an artificial gastrointestinal tract. Lactobacillus rhamnosus GG (LGG) is one of the most popular probiotics used in foods, functional foods, and supplements. The efficacy and functionality of LGG strongly depend on the dose taken and residence time in the gastrointestinal tract. In this study, the Wyndmoor scientists evaluated the growth and colonization of the probiotic using a simulator of the human intestinal microbial ecosystem and human fecal samples of three donors. The results revealed that LGG was actively replicating but could not permanently colonize in a mature gut microbial community, as confirmed by the differences between mechanistic calculation and qPCR measurement and by peak-to-trough analysis based on shotgun DNA sequencing results. The inclusion of LGG promoted or suppressed the growth of specific bacterial genera, yet the bacterial community was able to maintain homeostasis during LGG’s transient colonization. As determined by GC-MS and LC-MS/MS, the inclusion of LGG had a limited impact on bile acids conversion and the uptake and production of amino acids. LGG influenced the production of acetic, propanoic, and butanoic acids. These significant effects were detected on days 2-8 after inoculation and reduced as the probiotic was removed from the system. Furthermore, the impact of LGG on the gut microbial structure and the metabolites were donor-dependent and differentiated between colonic region and phase. The results of this research provide helpful information for scientists and enterprises in the fields of food, functional foods, and medicine, both in academia and industry, to develop novel applications of this probiotic. These results were summarized in a research paper submitted for review. Transcriptomics analysis of tomato treated with carbon dioxide. Tomatoes are a perishable and seasonal fruit with a high economic impact. Carbon dioxide, among several other reagents, is used to extend the shelf-life and preserve the quality of tomatoes during refrigeration or packaging. To obtain insight on CO2 stress during tomato ripening, tomatoes at the late green mature stage were conditioned with one of two CO2 delivery methods, 5% CO2 for 14 days (T1) or 100% CO2 for 3 hr (T2). Conventional physical and chemical characterization found that CO2 induced by either T1 or T2 delayed tomato ripening in terms of color change, firmness, and carbohydrate dissolution. However, T1 had longer-lasting effects. Furthermore, ethylene production was suppressed by CO2 in T1 and promoted in T2. These physical observations were further evaluated by RNA-Seq analysis at the whole genome level, including genes involved in ethylene synthesis, signal transduction, and carotenoid biosynthesis. Transcriptomics analysis revealed that the introduction of CO2 by method T1 downregulated genes related to fruit ripening, and the introduction of CO2 by method T2 upregulated the gene encoding for ACS6, the enzyme responsible for S1 ethylene synthesis, even though there was a large amount of ethylene present. Quantitative real-time PCR assays (qRT-PCR) were used for validation, which substantiated the RNA-Seq data. The results of the present research provide insight on gene regulation by CO2 during tomato ripening at the whole genome level. The research results were summarized in a manuscript, “Transcriptomic analysis on the regulation of tomato ripening by carbon dioxide,” and an invention disclosure was submitted. Gut microbial enzymes mediate triclosan-induced inflammation. Metabolic transformations play critical roles in the toxicities of environmental pollutants and toxicants. While previous research has focused on metabolism by host tissues, the biotransformations of environmental compounds by the gut microbiome are understudied. The Wyndmoor scientists with collaborators in the U. of Massachusetts, Stanford U., U. of N. Carolina, Hong Kong Baptist University, Brigham and Women's Hospital, Boston, and University of Shanghai for Science and Technology explored the specific gut microbial enzymes that metabolize the environmental compound triclosan (TCS) and revealed the functional impact of microbial metabolism of TCS on gut toxicity. After TCS exposure in both mice and humans, the intestines have a distinct metabolic profile of TCS compared to other tissues. Using in vitro, in fimo, and in vivo approaches, it was shown that the unique metabolic profile of TCS in the gut is mediated by actions of microbial ß-glucuronidase (GUS) enzymes, notably the Loop-1 GUS orthologs, present in the commensal microbiota. These enzymes mediated metabolic activation of TCS in the colon, contributed to the adverse effects of this environmental toxin, and could lead to therapeutic interventions for chronic gut diseases. The research results were summarized as a manuscript, "Specific Gut Microbial Enzymes Drive Colitis Promotion by Triclosan," and submitted to Nature: Metabolism (Log. No. 380936). The manuscript is under review.


Review Publications
Arserim-Ucar, K., Korel, F., Liu, L.S., Yam, K.L. 2020. Characterization of bacterial cellulose nanocrystals: Effect of acid treatments and neutralization. Food Chemistry. 1-23.
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.
Muhidinov, Z., Ikromi, K., Jonmurodov, A., Nasriddinov, A., Usmanova, S.R.,Bobokalonov, J., Strahan, G.D., Liu, L.S. 2021. Characterization of pectin obtained by conventional and innovative methods. International Journal of Biological Macromolecules. 183:2227-2237.
Yang, D., Hu, Z., Gao, J., Zheng, Z., Wang, W., Liu, J., Firrman, J., Ren, D. 2021. Probiotic effects of Lactobacillus fermentum I631 and Lactobacillus plantarum ZY08 on hypercholesteremic golden hamsters. Frontiers in Nutrition. 8:705763.