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ARS Home Northeast Area Wyndmoor, Pennsylvania Eastern Regional Research Center Dairy and Functional Foods Research Research





- Liu, Linshu
- Firrman, Jenni
- Mahalak, Karley
- Narrowe, Adrienne
- 2023
- 2022
- 2021
- Publications
- Product Quality and New Uses (306)

Research Project: In vitro Human Gut System: Interactions Between Diet, Food Processing, and Microbiota **Location:
                            Dairy and Functional Foods Research** 2022 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**
                                

Subobjective 1b: Lactose as a modifier of the human gut microbiota. Following consumption of milk, lactose, a disaccharide of glucose and galactose, is hydrolyzed and absorbed in the upper gastrointestinal tract. However, this is not always absolute, and some lactose will enter the colon where the gut microbiota will hydrolyze lactose and produce metabolic byproducts. In this project, ARS scientists in Wyndmoor, Pennsylvania, studied the impact of lactose on the gut microbiota of healthy adults, using an in vitro strategy where fecal samples harvested from eighteen donors were cultured anaerobically over 24 hours with and without lactose. Metagenomic analysis found that the addition of lactose decreased richness and evenness, while enhancing prevalence of the beta-galactosidase gene within the community. Taxonomically, lactose treatment decreased abundance of Bacteroidaceae, and increased lactic acid bacteria, Lactobacillaceae, Enterococcaceae, and Streptococcaceae, and the probiotic taxa Bifidobacterium. This corresponded with an increase in lactate utilizers, Veillonellaceae. The changes to community structure coincided with an increase in total short chain fatty acids (SCFAs), specifically acetate, and lactate. The results of this study demonstrated that lactose could mediate the gut microbiota structure and function in healthy adults in a donor-independent manner and provided insight into how dietary milk consumption may promote human health through modifications of the gut microbiome. A research paper is in preparation. 

Subobjective 2C: Effects of Fructooligosaccharide and 2’-Fucosyllactose on the human gut microbiome. Fructooligosaccharide (FOS) is a carbohydrate found in many vegetables that is often used as a prebiotic. 2’-Fucosyllactose (2’-FL) is a well-known human milk oligosaccharide (HMO) that promotes a healthy gut microbiota. FOS and 2’-FL have a previously demonstrated the ability to enhance levels of Bifidobacterium. In this study, ARS scientists in Wyndmoor, Pennsylvania, used a 24-hour in vitro culturing method to compare the effects of 2’-FL and FOS, separately, on different age groups ranging from infants to elder adults. The cultured communities were subject to 16s rRNA sequencing to determine structure and LC-MS/MS analysis to measure short-chain fatty acid (SCFA) levels, which is an indicator of community function. qPCR analysis targeting Bifidobacterium was applied to measure the effects of FOS and 2’-FL on this taxon. The results of these studies demonstrate how age may play a role in the outcome of treatment and/or consumption of FOS or 2’-FL. Two manuscripts are in preparation.

Subobjective 2C: Effects of chlorine on the gut microbiota in mouse models. Safe drinking water is often achieved through the use of chlorination, in which a low level of chlorine is added in order to deter growth of disease-causing microbes. Water chlorination has been a standard process in the United States for over a century, and chlorinated drinking water is considered as safe for human consumption. However, the gut microbiota that resides within the gastrointestinal tract is also subject to chlorinated drinking water, yet whether or not the presence of chlorine will affect this community was unclear. An ARS scientist in Wyndmoor, Pennsylvania studied the effect of chlorinated drinking water on the gut microbiota in vivo using a mouse model. Post weaning, male and female B6 mice were separated into two groups, one of which was provided chlorine-free water and one was provided water containing 4 ppm chlorine for four weeks. Each week, feces was harvested from both groups of mice, and at the end of the experiment, all mice were euthanized and their cecal content removed. The results of this study compare the microbial composition between mice provided non-chlorinated water to those provided chlorinated water and show the potential impact that chlorinated water may have on this community. All animal experiments were completed, the analysis of mice’s gut microbiome and metabolome is ongoing.   

Subobjective 2C: Difference in the effect on gut microbiota between soluble and insoluble fibers. Many soluble fibers are used in the food industry as thickeners and stabilizers, and they also alter the fermentation process and microbial metabolism. Insoluble fiber can improve bowel-related health problems, like constipation, hemorrhoids, and fecal incontinence. However, in comparison to soluble fibers, the interactions of insoluble fibers with the gut microbiota are less well-known. ARS scientists in Wyndmoor, Pennsylvania, conducted comparative in vitro research on the impact of soluble versus insoluble fibers on the gut microbiota, using a stable gut microbial culture established within TWINSHIME®. The research evaluates the dynamic change in the gut microbial community induced by soluble or insoluble rice fibers, bile salt conversion, and short chain fatty acids produced. The bench fermentation experiments were completed, and the analysis of the microbiome and metabolome is in process. 
 
Within-individual comparison of the development of the pig gut microbiome in vivo versus that developed in SHIME from fecal samples. TWINSHIME® made by ProDigest (Belgium) is used by ARS scientists in Wyndmoor, Pennsylvania, for in vitro studies evaluating the response of the human gut microbial community to foods and food components. Although it incorporates many elements of the human gastrointestinal tract, a precise measure of the extent of the similarity or dissimilarity between the human gut and the SHIME remains unknown. Therefore, we collaborated with ProDigest to conduct an in vivo versus in vitro study, comparing data derived from an animal model to data derived from the TWINSHIME®. Twelve pigs were used in this study. Pigs were fed with standardized SHIME feed for 2 weeks, and on day 14, each pigs’ fecal samples were collected separately. Immediately after collection, the pigs were slaughtered following European Union's regulation of Animal Welfare, the gut microbial samples of each pig were harvested along the gastrointestinal tract, separated by region and phase (lumen versus mucosa). Both the fecal and the gut samples were stored at -78 °C anaerobically. The TWINSHIME® was then inoculated with the fecal samples collected from each pig and operated under standard conditions for 14 days. Samples were taken from the luminal and mucin phases of bioreactors representing different colonic regions. To facilitate direct comparison of the gut microbial structure, samples collected from pig’s gastrointestinal tract (GIT) and from the TWINSHIME® were analyzed for the gut microbiota using 16S rRNA amplicons, and SCFA content. Status: The in vivo experiments were completed, the in vitro experiments will follow at a later date. 
 
Subobjective 2D: Using in vitro food digestate on intestinal cells in culture. Diet is strongly correlated with individual health outcomes. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with researchers from the Perelman School of Medicine at the University of Pennsylvania, developed a project to test the hypothesis that digestate from healthy and unhealthy diets may induce different cellular responses in the epithelial cells of the intestinal tract. Test meals, identical to those given to healthy human subjects as part of an ongoing Penn Medicine dietary study, were digested using a previously published in vitro protocol that mimics the oral, gastric, and small intestinal phases of digestion. The resulting digestates contained still active digestive enzymes and high bile acid concentrations which were highly toxic to Caco-2 monolayers. A purification technique was developed to allow digestates to be added to cells with no toxicity. Experiments are ongoing to explore the differential effects of the test meals on intestinal cells. Future plans include the addition of a bacterial fermentation step to mimic the colonic phase of digestion which may require the addition of yet more purification steps. Work is also underway to use primary intestinal epithelial cells instead of cancer cell lines. We are collecting data for a research publication and grant proposal now.  

Subobjective 1B: Disruption of intestinal oxygen dynamics during acute colitis alters the gut microbiome. The juxtaposition of oxygenated intestinal colonic tissue with an anerobic luminal environment is a fundamentally important relationship that is altered in the setting of intestinal injury. ARS scientists in Wyndmoor, Pennsylvania, in collaboration with scientists at University of Pennsylvania (UPENN) and the Children's Hospital of the University of Pennsylvania (CHOP), performed an in vivo/in vitro examination on the effect of physiological relevant levels of oxygen on the colon gut microbiota. Using phosphorescence quenching to quantify both intestinal tissue and luminal oxygenation in real time, we show that intestinal injury induced by dextran sodium sulfate (DSS) colitis reduces intestinal tissue oxygenation and increases the flux of oxygen into the gut lumen. By characterizing the composition of the gut microbiome in both DSS colitis as well as in an in vitro bioreactor containing a stable human fecal community exposed to microaerobic levels of oxygen, we provide evidence that the increased flux of oxygen augments glycan degrading bacterial taxa rich in glycoside hydrolases known to inhabit gut mucosal surface. Continued disruption of the intestinal mucus barrier through such a mechanism may help perpetuation the intestinal inflammatory process. The experiments have been performed and a manuscript is in preparation. **Accomplishments**
                                

1. Capsaicin, the bioactive compound in chili peppers, changes the gut microbial community in vitro. Capsaicin has been shown in previous studies to have a beneficial effect on human health, including protective effects against obesity. These beneficial health effects are often attributed to changes that capsaicin causes to the gut microbial community in vivo. ARS scientists in Wyndmoor, Pennsylvania, designed and executed an in vitro experiment to determine what effect capsaicin has on the in vitro human gut microbial community. It was found that capsaicin increased gut microbial diversity and the concentration of specific short-chain fatty acids. The results indicated that the beneficial health effects   of capsaicin are likely due to the changes that capsaicin induces in the gut microbial community. However, the specific changes in the gut microbial community, as well as the extent of these changes, are dependent on the individual. 2. A combination of human physiology and microbial metabolism produces a pH gradient along the colon. It is a critical factor for the development of the gut microbial community, a known contributor to human health. Changes to pH within microenvironments of the colon are often associated with disease and disease progression, however, the effect of pH change on the gut microbiota was unclear. To determine the ecological impact of environmental pH on the gut microbiota, ARS scientists in Wyndmoor, Pennsylvania, applied an in vitro model designed to simulate the human colon. The results demonstrated that both lowering and raising pH elicited significant changes to community structure in a donor-independent manner. Additionally, a more alkaline pH stimulated short chain fatty acid production, while these metabolites were reduced in the acidic environment. These results demonstrate that environmental pH is a critical parameter that can modulate gut bacterial community structure and function, which play an important role in human health and disease prevention. **Review Publications**
                            *Scarino Lemons, J.M., Liu, L.S. 2022. Chewing the fat with microbes: lipid crosstalk in the gut. Nutrients. 14(3):573. https://doi.org/10.3390/nu14030573.*
                                *Mahalak, K.K., Bobokalonov, J., Firrman, J., Williams, R., Evans, B., Fanelli, B., Soares, J., Liu, L.S., Kobori, M. 2022. Analysis of the ability of capsaicin to modulate the human gut microbiota in vitro. Nutrients. https://doi.org/10.3390/nu14061283.*
                                *Zhang, G., Redinbo, M., Cai, Z., Xiao, H., Liu, L.S., Gibbons, J., Kim, D., Minter, L., Panigrahy, A., Yang, J. 2022. Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract. Nature. https://doi.org/10.1038/s41467-021-27762-y.*


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