Location: Dairy and Functional Foods Research
2023 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
The effect of acylcarnitines on the gut microbiota in inflammatory bowel disease. Scientists have previously observed that the levels of the fatty acid intermediary metabolites, acylcarnitines, are elevated in the stools of patients with inflammatory bowel disease (IBD) and that this increase positively correlates with disordered gut microbiomes. ARS scientists, in collaboration with researchers at the University of Pennsylvania and the Children’s Hospital of Philadelphia, have extended this finding by establishing that acylcarnitines are also consumed by the microbiome. In particular, acylcarnitines appear to be able to support the growth of potentially pathogenic Enterobacteriaceae like E. coli which may contribute to pathogenesis of IBD. This research has resulted in 1 submitted manuscript and is related to Subobjective 2d.
In vivo study on Triclosan’s impact on the gut microbiota and gut health. Triclosan (TCS) is a general antimicrobial, residual TCS is found in water, soil, foods, and can cause severity of colitis and gut dysbiosis in disease models. Our previous research on SHIME found that residue TCS can be removed in 2 weeks after stopping the use, the dysbiosis of the gut microbiota caused by TCS was 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 in vitro results could be reproduced in a mammalian mouse model. All experiments were completed data analysis is in progress. This will result in 1 manuscript. This research is related to Subobjective 2c.
Insoluble and soluble rice bran fiber supplementation have different effects on the gut microbiota. However, how the difference in fiber types impact on the gut microbial composition and metabolism is still not clear. ARS scientists in Wyndmoor, Pennsylvania, conducted comparative research using an in vitro gut microbiota model established using the TWINSHIME® system. All experiment and data analysis were done, a manuscript is in preparation. This research is related to Subobjective 2C.
Ivermectin (IVM) is used to combat a broad spectrum of intestinal helminthic parasites and ectoparasites, and is also used for the treatment of filarial diseases and in malarial control, has been demonstrated as an effective, safe, and affordable medicine. A trend of the current research is to search for the re-purposing uses of IVM. That is both scientifically and economically meaningful, since it is costly and time-consumed to develop a new medicine. As part of consideration of its new applications, it is important that a short-term IVM administration should not largely or persistently alter the composition of the host microbial community. ARS scientists in Wyndmoor, Pennsylvania, tested IVM’s impact on gut microbial activity, composition and function in the context of fiber-degrading ability. A manuscript is in preparation.
Probiotics are beneficial to human health, are widely used as dietary supplements and in the functional food industry. The interactions of the probiotics and foods, food components and metabolites have the potential to affect their availability and activity, yet it is less, and in some case, even no studied. Using Lactobacillus rhamnosus GG (LGG) as a model probiotic, ARS scientists in Wyndmoor, Pennsylvania, cultured LGG in the presences of food components and/or metabolite. The growth profiles and the transcriptomics of LGG activity across the treatment were investigated and results are in analysis. Two manuscripts will be submitted. This research is related to Subobjective 2C.
Development of an in vitro small intestine gut microbiota model. A small intestine gut microbiota community was developed in vitro using ileostomy samples as the inoculum, and the mature community was analyzed for composition and function under anoxic and oxic conditions. One manuscript is in preparation, and one has been submitted. This research is related to Objectives 1A and 2A.
Lactose as a modifier of the small intestine gut microbiota. The effect of the milk component lactose on the small intestine gut microbiota was analyzed in vitro through detection of structural and functional changes to the community that occurred upon the administration of lactose. The results will show how lactose may impact the small intestine gut microbiota, which will provide insight into how dietary milk consumption may promote human health through modifications of the gut microbiome. This research is related to Objective 1A.
TWINSHIME® evaluation by comparing with correlative in vivo model. A gut microbial community was established in a Twin Simulator of Human Intestinal Microbial Ecosystem using pig fecal samples and compared with that obtained from gastrointestinal tract of the same pig in terms of composition and metabolites. Data is in analysis. A manuscript is planned to submit by the end of this calendar year. This research is related to Objectives 1 and 2.
Investigating the effect of eleven traditional Chinese remedies on the structure and function human gut microbiota. The effects of eleven traditional Chinese remedies on the structure and function of human gut microbiota were investigated using SIFR® technology. Their findings suggest that the gut microbiota is affected by the phytochemicals present in the various extracts and that some of the beneficial effects of these extracts may, in part, be due to their effect on the microbiome. Manuscripts are in preparation.
Accomplishments
1. Following consumption of milk, lactose, a disaccharide of glucose and galactose, is hydrolyzed and absorbed in the upper gastrointestinal tract. However, hydrolysis and absorption are not always absolute, and some lactose will enter the colon where the gut microbiota can consume lactose. Yet the effect of lactose on the gut microbial community, which is a known contributor to human health, was unclear. To address this gap in knowledge, ARS scientists in Wyndmoor, Pennsylvania, applied a short-term, in vitro culturing strategy where the gut microbiota of eighteen donors were cultured with and without lactose, and the data compiled to identify donor-independent responses to treatment. The results found that lactose mediated the gut microbiota in a donor-independent manner that was consistent with other described prebiotics, increasing levels of the health-associated microbe Bifidobacterium and stimulating short chain fatty acid production. Together, these results provided further insight into how dietary milk consumption promotes human health through modifications of the gut microbiome.
2. Lacticaseibacillus rhamnosus strain GG (LGG) is commonly used and sold as a probiotic, and there are health benefits associated with its presence in the gut microbial community. However, it is not well known how LGG travels through the gut, how long a treatment dose may last, and what other benefits might be conferred due to its use. Aiming at resolving this issue, ARS scientists in Wyndmoor, Pennsylvania, designed and performed an in vitro experiment to understand the ability of LGG to persist within an established gut microbial community and determine how LGG can change the function of said community. This work found that LGG persists within the in vitro gut microbial community for at least 7 days after introduction, with little to no additional effects and that the presence of LGG within the gut promotes the production of tryptophan-pathway metabolites such as indole propionic acid. These findings were true across multiple donor communities, indicating that the health benefits associated with LGG are likely due to its ability to engraft into the gut microbial community and adjust metabolic functions.
3. Fructooligosaccharides (FOS) are carbohydrates found in many vegetables (chicory root, onions, garlic, etc.) and are often used as a prebiotic. FOS has been previously demonstrated to increase Bifidobacterium within the gut microbiota, a beneficial microbe that is often more prevalent in children and young people. In this study, ARS scientists in Wyndmoor, Pennsylvania, collaborated with an international research team to use a new short-term culturing method to understand how FOS changes the gut microbiota of different age groups. In this study, donors ranged in age from 25-70, and were separated into young adult, adult, and older adult age groups. The results of this study found age-specific changes in the gut microbiota in response to FOS treatment, demonstrating that different members of Bifidobacteria become more critical in metabolic pathways related to the breakdown of carbohydrates at different age groups. These results demonstrate that FOS can be a useful prebiotic at any age group.
4. Pectins are plant polysaccharides consumed as part of a diet containing fruits and vegetables. Inside the gastrointestinal tract, pectin cannot be metabolized by the mammalian cells but is fermented by the gut microbiota in the colon with the subsequent release of short-chain fatty acids. Previous reports on the prebiotic effects of pectin have produced inconsistent results, most likely due to differences in the pectin chemical structure. To address this gap in knowledge, ARS scientists in Wyndmoor, Pennsylvania, applied an in vitro model to evaluate the effects of two structurally different lemon pectins on the gut microbiota of two donors. The results found that both lemon pectins were able to alter community structure and enhance levels of short-chain fatty acids. Together, these data provide valuable information linking chemical structure of pectin to its effect on the gut microbiota structure and function, which is important to understanding its prebiotic potential.
Review Publications
Mahalak, K.K., Firrman, J., Narrowe, A.B., Hu, W., Bittinger, K., Moustafa, A., Liu, L.S. 2023. Fructooligosaccharides (FOS) differentially modifies the in vitro gut microbiota in an age-dependent manner. Frontiers in Nutrition. https://doi.org/10.3389/fnut.2022.1058910.
Firrman, J., Liu, L.S., Mahalak, K.K., Tu, V., Tanes, C., Bittinger, K., Bobokalonov, J., Mattei, L., Zhang, H., Van Den Abeele, P. 2022. The impact of environmental pH on the gut microbiota community structure and short chain fatty acid production. FEMS Microbiology Ecology. 98(5). https://doi.org/10.1093/femsec/fiac038.
Mahalak, K.K., Firrman, J., Bobokalonov, J., Narrowe, A.B., Bittinger, K., Daniel, S., Tanes, C., Mattei, L., Zeng, W., Soares, J.W., Kobori, M., Scarino Lemons, J.M., Tomasula, M.M., Liu, L.S. 2022. Persistence of the probiotic Lacticaseibacillus rhamnosus strain GG (LGG) in an in vitro model of the gut microbiome. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms232112973.
Liu, L.S., Narrowe, A.B., Firrman, J., Mahalak, K.K., Bobokalonov, J., Scarino Lemons, J.M., Bittinger, K., Daniel, S., Tanes, C., Mattei, L., Friendman, E., Soares, J.W., Masuko, K., Zeng, W., Tomasula, M.M. 2023. Lacticaseibacillus rhamnosus strain GG (LGG) regulate gut microbial metabolites, an in vitro study using three mature human gut microbial cultures in a simulator of human intestinal microbial ecosystem (SHIME). Foods. https://doi.org/10.3390/foods12112105.
