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

Location: Dairy and Functional Foods Research

2024 Annual Report

Objectives
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.

Approach
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
An in vitro model of the small intestinal microbiota: The small intestinal microbiota has emerged as an important contributor to nutrient utilization, fueling the need for in vitro platforms to elucidate the functional role of this community. Here, ARS scientists in collaboration with the University of Pennsylvania, have developed an in vitro model of the small intestinal microbiota (SIM). Shotgun sequencing combined with metabolomics found that the interindividual variability between donors was maintained in vitro, with growth of typical taxa associated with the small intestine, and production of acetic acid with less propanoic acid was divergent from the function of the colon gut microbiota. These results demonstrate that the SIM model effectively recapitulates the in vivo small intestinal gut microbiota. All experiments for this project have been completed, the data is currently being analyzed. This research is related to Objectives 1A and 2A. Digestion models: in vitro vs. in vivo: The use of in vitro simulation for digestion studies that the resultant data would be the same as or close to those obtained from in vivo studies. To evaluate this, ARS scientists in Wyndmoor, Pennsylvania, inoculated fecal samples of experimental pigs in SPIME and cultured until a steady state was reached. The microbial composition and metabolites generated in vitro were compared with those harvested from the gastrointestinal tracts of the same pigs euthanized right after feces collection. All experiments and data analysis were completed. This research relates to Objectives 1 and 2. Effect of high fiber and polyphenol content nutrition bars on the human gut microbiota in vitro: High levels of dietary fiber intake are associated with a healthy gut microbial community. Polyphenols are beneficial to human health due to their effects on the gut microbiota and anti-inflammatory properties. A nutrition bar with multiple types of both dietary fiber and polyphenols from multiple plant sources is of interest to the military for use by warfighters to maintain gut health and reduce instances of stomach illnesses in deployment situations. In collaboration with scientists at the U.S. Army Combat Capabilities Development Command Soldier Center, ARS scientists tested the effects of the components of this bar for 1 week, using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®). Bench experiments are complete, and analysis is ongoing. This research is related to Subobjective 2c. In vivo study on Triclosan’s impact on the gut microbiota and gut health. Triclosan (TCS) is a general antimicrobial used in more than 2000 consumer products, and residual TCS is found in water, soil, and foods. TCS can enter the gastrointestinal tract and increase severity of colitis and gut dysbiosis in disease models. Our previous work on this topic added TCS to the in vitro gut microbiota using the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) and discovered that after a washout period of 2 weeks, the TCS-caused dysbiosis of the gut microbiota recovered into a functional community. Here, ARS scientists in Wyndmoor, Pennsylvania, in collaboration with the scientists of Perelman School of Medicine at the University of Pennsylvania, designed and implemented a study to determine whether our results could be reproduced in a mammalian mouse model. Analysis has been completed. This research is related to Subobjective 2c. Age as a determining factor for the response of the gut microbiota to the prebiotic 2’fucosyllactose (2’FL): The gut microbiota evolves in concordance with its host over a lifetime, yet it was unclear how age may affect the response of the gut microbiota to treatment with a prebiotic, such as 2’FL. Here, ARS scientists in collaboration with Cryptobiotix (Ghent, Belgium), examined the effect of 2’FL on the gut microbiota of humans spanning infancy to older adults (0-70y) using an ex vivo platform. Metagenomic data and mass spectrometry analysis found that age-specific communities clustered together and there was an age-related difference in the species of Bifidobacterium affected and corresponding cross-feeding interactions that occurred. These results highlight the importance of age on the outcome of prebiotic administration. All experiments have been completed; the data is currently being analyzed. This research is related to Objectives 1B and 2B. Effect of Allyl Isothiocyanate (AITC) on the human gut microbiota: AITC is a sulfur-containing compound in brassica plants that has anti-carcinogenic and anti-bacterial activity; and has reduced colitis in mouse models. Yet, whether these effects are related to changes in the gut microbiota caused by AITC is unknown. ARS scientists, with industry partner Cryptobiotix, used short-term, ex vivo culturing methods to find the impact of AITC on the gut microbiota of age groups ranging from infants to adults over 60 years old. ARS scientists also used the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) to find long-term effects on the gut microbiota. This research found that AITC takes over 48 hours to impact the gut microbial community globally but does make small changes to the gut microbiota, and increase butyrate production, over 24 hours in adults over 60. This research is related to Objectives 1B and 2B. The effect of lactose on the small intestinal gut microbiota: Lactose is a unique component of milk consumed as part of a diet containing milk products. Lactose can by hydrolyzed and absorbed by mammalian cells, however, many of the microbes that reside within the small intestine can also utilize lactose as a source of nutrition; Yet it was unclear if lactose may promote growth of specific microbes within the small intestine and to what extent. Here, ARS scientists in Wyndmoor, Pennsylvania, studied the impact of lactose on the small intestine gut microbiota using an in vitro model. The culturing experiments have been completed and results of metagenomic sequencing and functional output are currently being evaluated. Together, the results will show how lactose impacts the small intestine gut microbiota, providing insight into how dietary milk consumption promotes human health through modifications of the gut microbiome. This research is related to Objective 1A. The impact of chlorinated water on the gut microbiota: Water chlorination deters growth of microbes and decreases prevalence of communicable disease. Toxicological studies have found chlorinated water safe for human consumption, yet the effect of chlorine in water on the gut microbiome was unclear. Here, ARS scientists in collaboration with University of Indiana, analyzed the impact of chlorinated water on mice. The results found that chlorinated water had no impact on community diversity, only a minimal impact on the community structure, and was associated with a decrease in several virulence factors and genes involved in Vitamin B12 biosynthesis. Taken together, the results demonstrated that the gut microbiota of mice were resilient to low levels of chlorine with no changes to diversity or evidence of major community restructuring. The experiments have been completed and the manuscript is currently under review. This research is related to Objectives 1B and 2B. Disruption of intestinal oxygen dynamics during acute colitis alters the gut microbiome. In the colon, there is an anatomical delineation between the human cells lined with mucus and the lumen interior. During disease states, injury to the human cells can disrupt this stratification and allow oxygen to leak out from the human cells into the lumen, yet it was unclear how this may affect the gut microbiome and to what extent. Here, ARS scientists in collaboration with University of Pennsylvania, found that injury to the human cells did result in oxygen entering the lumen, and that enhanced levels of mucus-consuming taxa that may infiltrate the mucous layer and further worsen intestinal injury. Together, these results described the intereffects of intestinal injury and the gut microbiota, important to understanding the etiology of colon disease. The experiments have been completed and the manuscript is currently under review. This research is related to Objectives 1B and 2B. Cheese is a carrier of beneficial bacteria. The intestinal microbiota responds to cheese digestion through changes in community structure and metabolite production. To evaluate how the bacteria within the cheese matrix influence protein and lipid digestion, ARS scientists in Wyndmoor, Pennsylvania, conducted in vitro research on cheese digestion in a Simulator of Human Intestinal Microbial Ecosystem (SHIME). Stable gut microbial communities representing either the small or large intestine microbiota were established separately. Cheese incorporated with probiotics was added and the dynamics of its digestion were monitored by metabolome and bacterial composition analysis. All bench experiments were completed, data was analyzed, 2 manuscripts are in preparation. This research is related to Subobjectives 2A and 2B. Ensuring global food security for this growing population is an ongoing challenge that will likely require increased sustainable food production and reduced food loss via processing techniques that increase safety and shelf-life. Researchers at the ERRC in Wyndmoor, Pennsylvania, are exploring the impact of butylated hydroxyanisole (BHA), a food preservative whose safety has recently been called into question, on the gut microbiome. Their results suggest that at doses higher than those likely to be consumed by humans BHA does not significantly affect the microbial community of the gut in either a beneficial or detrimental way. This research is related to Subobjectives 2A and 2B.

Accomplishments
1. Acylcarnitines can support the growth of Escherichia coli (E. coli) providing a causal link between their elevation and inflammatory bowel disease (IBD) pathogenesis. Scientists have previously observed that the levels of the fatty acid intermediary metabolites, acylcarnitines, are elevated in the stools of patients with dysbiotic IBD. Dysbiosis is characterized by an accumulation of potentially pathogenic bacteria and a reduction in beneficial bacteria. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with researchers at the University of Pennsylvania and the Children’s Hospital of Philadelphia, confirmed these previous observations, but in a longitudinal pediatric cohort study. They also showed, using germ-free animal models and bacterial culturing studies, that these metabolites originate from the host and are capable of supporting the growth of the bacteria that drive dysbiosis.

2. The presence of oxygen in the small intestine inhibits deconjugation of bile acids by the gut microbiota. Bile acids play a central role in the digestion and absorption of food in the small intestine. Bile acid function can be disrupted by the gut microbiota via deconjugation followed by the conversion from primary to secondary species. Although this conversion reaction is performed by the microbiota in the colon quite rapidly, it is not commonly observed in the small intestine. It was hypothesized that the lack of bile acid conversion in the small intestine may be due to the composition of the microbiota or the presence of oxygen in the small intestine environment. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with University of Pennsylvania, modeled the small intestinal microbiota in vitro and quantified levels of bile acids, monitoring their deconjugation and conversion using chemical analysis. The results found that infusion of oxygen reduced deconjugation of bile acids, in part, through the expansion of microbes unable to perform this reaction. These results provide unprecedented evidence that the oxygenated environment of the small intestine plays an important role in the maintenance of normal digestive physiology.

3. Soluble and insoluble fiber from Rice Bran causes positive changes in the gut microbiota community. A lack of fiber consumption in American is likely to be a major contributing factor to the increase in many digestive disorders, including irritable bowel diseases. Rice bran is a high-fiber by-product of the process used to create white rice with the potential to be used as a dietary supplement. Fiber is digested in the colon by the gut microbiota, and this process produces beneficial compounds that promote gut and immune health. However, there are multiple types of fiber, including soluble and insoluble fiber, and their different effects on the gut microbiota are not well understood. ARS Scientists in Wyndmoor, Pennsylvania, used an in vitro model of the human colon gut microbiota to understand how microbial communities develop differently following regular supplementation with either insoluble or soluble fibers from rice bran. The results demonstrated that both fiber types increased beneficial bacteria, specifically Bifidobacterium and Lachnospiraceae taxa in the population as well as short-chain fatty acids, specifically butyrate. These results indicate that regular consumption of both soluble and insoluble rice bran fiber can increase the health of the gut microbiome, revealing a new supplement that can play a beneficial role in human health as a supplement.

4. Disruption of the gut microbiota can lead to the outgrowth of opportunistic pathogens that contribute to disease, such as Klebsiella pneumonia (K. pneumonia) which is associated with infection. A desirable method to modulate growth of K. pneumonia would be through diet; however, detailed information on K. pneumonia’s nutrient requirements and how this may be altered within the colon environment during periods of nutrient limitations is unknown. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with University of Pennsylvania, detailed the role of carbohydrates and nitrogen on growth of K. pneumonia, and elucidated the response of K. pneumonia when either of these dietary components were limited. The results found that simple carbohydrates, and not nitrogen, were important to growth and survival of this pathogen, and that a diet containing fiber helped reduce the ability for K. pneumonia to colonize the colon. In conclusion, these results showed how dietary components may promote or hinder growth and colonization of the human pathogen K. pneumonia. This provided fundamental information required to formulate.

5. Ivermectin (IVM) is not harmful to the gut microbial ecosystem. IVM is an effective and affordable anti-parasitic medicine used for human and veterinary care. While further research on oral delivery of IVM has shown potential re-purposing applications in combating non-parasitic intestinal infections, information is not available regarding its effect on the homeostasis of the gut microbial ecosystem of healthy individuals, despite the need for such information for application and scientific regards. ARS scientists in Wyndmoor, Pennsylvania, tested IVM’s impact on gut microbial structure and functionality using DNA marker gene sequencing coupled with metabolomics. The Triple-SHIME in vitro platform was applied to provide conditions to examine the interaction between IVM and gut microbiota while eliminating the interplay between microbial and host cells that would occur in vivo. It was found that IVM is not expected to induce dysbiosis or yield adverse effects if administered to healthy adults with regular diet. This research benefits pharmaceutical industries and the public, with indirect impact on food industries.

6. Tomato seeds as health-promoting supplements. Tomato seeds are often discarded in tomato pomace, a byproduct of tomato processing, yet these seeds are known to contain an array of compounds with biological activity and prebiotic potential. Here, ARS scientists in Wyndmoor, Pennsylvania, tested extract from tomato seeds (TSE), acquired from pomace, for their ability to effect changes on the gut microbiota using an ex vivo strategy. The results found that TSE significantly enhanced levels of the beneficial bacteria Bifidobacteriaceae which corresponded with a significant increase in the release of health-associated microbial metabolites. Together, the results demonstrated that TSE has prebiotic potential by shaping the gut microbiota in a manner beneficial to human health. These findings provide a novel application for TSE harvested from tomato pomace and demonstrate the potential to further valorize tomato waste products.

7. Senna seed extracts disrupt the gut microbiome. Senna plants are found worldwide, and Senna leaf or seed preparations, such as Senna teas, are popular for their laxative effects. Despite extensive knowledge of how individual Senna components directly affect the human gastrointestinal tract, there is limited knowledge of how full Senna preparations affect the beneficial microorganisms living there. To address this question, ARS scientists in Wyndmoor, Pennsylvania, used short-term fecal incubations to test the effects of Senna seed extracts on the human gut microbiome. The results showed that Senna extracts in high concentrations had a disruptive effect on the gut microbiome, by specifically targeting members of a main bacterial group, the Bacteroidaceae. These results demonstrate that even well-accepted products may have unintended impacts on human health and nutrition via effects on the microbiome. The results of this research are beneficial to the public who may consume these products and to the scientific community interested in the potential for novel antimicrobial activity.

8. The beneficial effects of some herbs used in Traditional Chinese medicine may be partially attributable to their effect on the gut microbiome. There is a long history of utilizing plants for their medicinal properties. Three such herbs, Baizhu, Daqingye, and Hehuanhua, have been used for centuries to treat gastrointestinal illnesses, epidemics, and mental health issues, respectively. Given the strong links between the gut microbiome and human health and the known ability of plant polyphenols to exert changes on the gut microbiome, ARS scientists in Wyndmoor, Pennsylvania, in collaboration with researchers from Cryptobiotix, Ghent, Belgium, and the University of Maryland, asked whether the medicinal qualities of these herbs might be attributed to their impact on the gut. The impact of these herbs on the gut microbiota of six different donors was assessed via short-term incubation in Systemic Intestinal Fermentation Research (SIFR®) bioreactors. They found that some of the bacteria that increased in response to these herbs have previously been found to confer similar health benefits as the herbs themselves hinting that the microbes may mediate these effects.

9. Open data sharing provides additional value from research efforts. Studies of the human gut microbiome often seek to identify specific bacteria or groups of bacteria associated with health benefits. Gut microbiomes can vary greatly across individuals, yet these divergent microbiomes can be functionally equivalent. In contrast, even similar microbiomes can have very different functions making the identification of universally beneficial microbes a formidable challenge. Overcoming these challenges requires the use of large datasets that include samples from many individuals, making large-scale open data sharing critical to unlocking the potential found within the human gut microbiome. ARS scientists in Wyndmoor, Pennsylvania, performed studies on the effects of selected plant extracts on the human gut microbiome. From the DNA sequencing data used for these studies, the scientists additionally curated and made available a collection of 143 metagenome-assembled microbial genomes (MAGs) representing the gut microbiomes of six healthy adult human donors. By adding to the catalog of publicly available human gut microbiome data, this data resource ultimately benefits the public and scientists by enabling data reuse for further research into microbial variability and function across individuals.

Review Publications
Narrowe, A.B., Liu, L.S., Scarino Lemons, J.M., Firrman, J., Mahalak, K.K., Deyaert, S., Baudot, A., Van Den Abbeele, P. 2024. Metagenomes and metagenome-assembled genomes from ex vivo fecal incubations of six unique donors. Microbiology Resource Announcements. https://doi.org/10.1128/mra.00862-23.
Bobokalonov, J., Liu, Y., Mahalak, K.K., Firrman, J., Sheen, S., Zhou, S., Liu, L.S. 2023. Transcriptomic analysis on the regulation of tomato ripening by carbon dioxide. Sci. 5(3):26. https://doi.org/10.3390/sci5030026.
Narrowe, A.B., Scarino Lemons, J.M., Mahalak, K.K., Firrman, J., Van Den Abbeele, P., Baudot, A., Deyaert, S., Li, Y., Yu, L., Liu, L.S. 2024. Targeted remodeling of the human gut microbiome using Juemingzi (Senna seed extracts). Frontiers in Cellular and Infection Microbiology. 14. https://doi.org/10.3389/fcimb.2024.1296619.
Mahalak, K.K., Liu, L.S., Bobokalonov, J., Narrowe, A.B., Firrman, J., Bittinger, K., Hu, W., Jones, S.M., Mustafa, A.M. 2024. Supplementation with soluble or insoluble rice-bran fibers increases short-chain fatty acid producing bacteria in the gut microbiota in vitro. Frontiers in Nutrition. https://doi.org/10.3389/fnut.2024.1304045.
Scarino Lemons, J.M., Narrowe, A.B., Liu, L.S., Firrman, J., Mahalak, K.K., Van Den Abbeele, P., Baudot, A., Deyaert, S., Li, Y., Yu, L. 2023. Impact of Baizhu, Daqingye, and Hehuan extracts on the human gut microbiome. Frontiers in Cellular and Infection Microbiology. 13:1298392. https://doi.org/10.3389/fcimb.2023.1298392.
Liu, L.S., Mahalak, K.K., Bobokalonov, J.D., Narrowe, A.B., Firrman, J., Scarino Lemons, J.M., Bittinger, K., Hu, W., Jones, S.M., Mustafa, A.M. 2023. Impact of ivermectin on gut microbial ecosystem. International Journal of Molecular Sciences. 24:16125. https://doi.org/10.3390/ijms242216125.
Firrman, J., Narrowe, A.B., Liu, L.S., Mahalak, K.K., Scarino Lemons, J.M., Van Den Abbeele, P., Baudot, A., Deyaert, S., Li, Y., Yao, Y., Yu, L. 2024. Tomato seed extract promotes health of the gut microbiota and demonstrates a potential new way to valorize tomato waste. PLOS ONE. 19(4):e0301381. https://doi.org/10.1371/journal.pone.0301381.
Firrman, J., Friedman, E.S., Hecht, A., Strange, W.C., Narrowe, A.B., Mahalak, K.K., Wu, G.D., Liu, L.S. 2024. Preservation of conjugated primary bile acids by oxygenation of the small intestinal microbiota in vitro. mBio. 15(6). https://doi.org/10.1128/mbio.00943-24.
Muhidinov, Z., Bobokalonov, J., Kimatov, R., Rahmonov, E., Komilova, G., Sherova, Z., Liu, L.S. 2024. A new approach to the treatment of acute infection diseases with antibiotic-pectin formulae. The Journal of Infection in Developing Countries. 18(3):407-419.
Scarino Lemons, J.M., Conrad, M., Tanes, C., Chen, J., Friedman, E.S., Roggiana, M., Curry, D., Chau, L., Hecht, A.L., Harling, L., Vales, J., Kachelries, K.E., Baldassano, R.N., Goulian, M., Bittinger, K., Master, S.R., Liu, L.S., Wu, G.D. 2023. Enterobacteriaceae growth promotion by intestinal acylcarnitines, a biomarker of dysbiosis in inflammatory bowel disease. Cellular and Molecular Gastoenterology and Hepatology. 17(1):131-148. https://doi.org/10.1016/j.jcmgh.2023.09.005.
Hetch, A., Harling, L., Friedman, E., Tanes, C., Lee, J., Firrman, J., Hao, F., Tu, V., Liu, L.S., Patterson, A.D., Bittinger, K., Goulian, M., Wu, G. 2024. Dietary carbohydrates regulate intestinal colonization and dissemination of Klebsiella pneumoniae. Journal of Clinical Investigation. 134(9):e174726. https://doi.org/10.1172/JCI174726.