Summary of Study ST004478

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002825. The data can be accessed directly via it's Project DOI: 10.21228/M8H555 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004478
Study Title	Responsiveness to 2’FL and GOS by infant gut microbiota differs by history of individual 2’FL consumption
Study Summary	The study involved performing a secondary analysis on fecal samples from a randomized, controlled trial (RCT) of 2’FL-supplemented or un-supplemented formula to investigate the relationship between dietary exposure to 2’FL and the subsequent ability of fecal microbiota to metabolize 2’FL and galacto-oligosaccharides (GOS). Microbial metabolic response to these two prebiotics was measured using in vitro anaerobic fermentation with metabolomics read-out, including short-chain fatty acids (SCFA). Microbial diversity was characterized by using whole metagenome sequencing of raw stool. Exposure to 2’FL in the trial formula arm primed the growth and metabolism of the microbiota toward increased utilization of 2’FL (p=0.005), whereas utilization of GOS, a prebiotic to which the infants had no prior exposure, declined (p=0.18). Individuals with no dietary exposure to prebiotics resulted in their gut microbiota being less responsive to both 2’FL (p=0.05) and GOS (p=0.05). Conclusions: The response to 2’FL or GOS in baseline fermentation analysis was associated with differences in microbial beta-diversity, suggesting that individual responsiveness to specific prebiotics may be predicted by anaerobic fermentation of their stool samples. Continuous supplementation of prebiotic may prime the gut microbiota to more efficiently utilize this prebiotic.
Institute	University of Cincinnati College of Medicine
Last Name	Thorman
First Name	Alexander
Address	160 Panzeca Way 125 Kettering Lab Complex, Cincinnati, OH, 45220, USA
Email	thormaaw@ucmail.uc.edu
Phone	513-746-5121
Submit Date	2025-12-19
Raw Data Available	Yes
Raw Data File Type(s)	fid
Analysis Type Detail	NMR
Release Date	2026-01-06
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002825
Project DOI:	doi: 10.21228/M8H555
Project Title:	Responsiveness to 2’FL and GOS by infant gut microbiota differs by history of individual 2’FL consumption
Project Summary:	Background: The microbiota strongly influences human gut homeostasis and health through its metabolites. The metabolic products of the infant microbiota are strongly influenced by human milk or formula feeding. Human milk contains oligosaccharides (HMOS) that are prebiotic, promoting gut colonization by mutualists. Most human milk contains 2’-fucosyllactose (2’FL) as its principal oligosaccharide, but its abundance varies. The goal of this study was to evaluate the metabolites of cultured stool microbiota in the presence of different prebiotics as an approach to predict prebiotic responsiveness. Our studies find that The response to 2’FL or GOS in baseline fermentation analysis was associated with differences in microbial beta-diversity, suggesting that individual responsiveness to specific prebiotics may be predicted by anaerobic fermentation of their stool samples. Continuous supplementation of prebiotic may prime the gut microbiota to more efficiently utilize this prebiotic.
Institute:	University of Cincinnati College of Medicine
Last Name:	Thorman
First Name:	Alexander
Address:	160 Panzeca Way 125 Kettering Lab Complex, Cincinnati, OH, 45220, USA
Email:	thormaaw@ucmail.uc.edu
Phone:	513-746-5121

Subject:

Subject ID:	SU004654
Subject Type:	Bacteria
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Bacteria; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Time Point	treatment
SA532327	AM511	Fecal culture media	0hr	Blank
SA532328	AM501	Fecal culture media	0hr	Blank
SA532329	AM503	Fecal culture media	0hr	Blank
SA532330	AM505	Fecal culture media	0hr	Blank
SA532331	AM509	Fecal culture media	0hr	Blank
SA532332	AM507	Fecal culture media	0hr	Blank
SA532333	AM513	Fecal culture media	0hr	Blank
SA532334	AM515	Fecal culture media	0hr	Blank
SA532335	AM517	Fecal culture media	0hr	Blank
SA532336	AM519	Fecal culture media	0hr	Blank
SA532337	AM521	Fecal culture media	0hr	Blank
SA532338	AM237	Fecal culture media	0hr	No sugar
SA532339	AM201	Fecal culture media	0hr	No sugar
SA532340	AM217	Fecal culture media	0hr	No sugar
SA532341	AM218	Fecal culture media	0hr	No sugar
SA532342	AM219	Fecal culture media	0hr	No sugar
SA532343	AM235	Fecal culture media	0hr	No sugar
SA532344	AM236	Fecal culture media	0hr	No sugar
SA532345	AM272	Fecal culture media	0hr	No sugar
SA532346	AM253	Fecal culture media	0hr	No sugar
SA532347	AM254	Fecal culture media	0hr	No sugar
SA532348	AM255	Fecal culture media	0hr	No sugar
SA532349	AM002	Fecal culture media	0hr	No sugar
SA532350	AM271	Fecal culture media	0hr	No sugar
SA532351	AM199	Fecal culture media	0hr	No sugar
SA532352	AM273	Fecal culture media	0hr	No sugar
SA532353	AM200	Fecal culture media	0hr	No sugar
SA532354	AM163	Fecal culture media	0hr	No sugar
SA532355	AM183	Fecal culture media	0hr	No sugar
SA532356	AM129	Fecal culture media	0hr	No sugar
SA532357	AM093	Fecal culture media	0hr	No sugar
SA532358	AM109	Fecal culture media	0hr	No sugar
SA532359	AM110	Fecal culture media	0hr	No sugar
SA532360	AM111	Fecal culture media	0hr	No sugar
SA532361	AM127	Fecal culture media	0hr	No sugar
SA532362	AM128	Fecal culture media	0hr	No sugar
SA532363	AM145	Fecal culture media	0hr	No sugar
SA532364	AM182	Fecal culture media	0hr	No sugar
SA532365	AM146	Fecal culture media	0hr	No sugar
SA532366	AM147	Fecal culture media	0hr	No sugar
SA532367	AM290	Fecal culture media	0hr	No sugar
SA532368	AM164	Fecal culture media	0hr	No sugar
SA532369	AM165	Fecal culture media	0hr	No sugar
SA532370	AM181	Fecal culture media	0hr	No sugar
SA532371	AM289	Fecal culture media	0hr	No sugar
SA532372	AM326	Fecal culture media	0hr	No sugar
SA532373	AM291	Fecal culture media	0hr	No sugar
SA532374	AM440	Fecal culture media	0hr	No sugar
SA532375	AM410	Fecal culture media	0hr	No sugar
SA532376	AM411	Fecal culture media	0hr	No sugar
SA532377	AM424	Fecal culture media	0hr	No sugar
SA532378	AM425	Fecal culture media	0hr	No sugar
SA532379	AM426	Fecal culture media	0hr	No sugar
SA532380	AM439	Fecal culture media	0hr	No sugar
SA532381	AM441	Fecal culture media	0hr	No sugar
SA532382	AM396	Fecal culture media	0hr	No sugar
SA532383	AM457	Fecal culture media	0hr	No sugar
SA532384	AM458	Fecal culture media	0hr	No sugar
SA532385	AM459	Fecal culture media	0hr	No sugar
SA532386	AM475	Fecal culture media	0hr	No sugar
SA532387	AM476	Fecal culture media	0hr	No sugar
SA532388	AM477	Fecal culture media	0hr	No sugar
SA532389	AM409	Fecal culture media	0hr	No sugar
SA532390	AM395	Fecal culture media	0hr	No sugar
SA532391	AM307	Fecal culture media	0hr	No sugar
SA532392	AM344	Fecal culture media	0hr	No sugar
SA532393	AM308	Fecal culture media	0hr	No sugar
SA532394	AM309	Fecal culture media	0hr	No sugar
SA532395	AM325	Fecal culture media	0hr	No sugar
SA532396	AM091	Fecal culture media	0hr	No sugar
SA532397	AM327	Fecal culture media	0hr	No sugar
SA532398	AM343	Fecal culture media	0hr	No sugar
SA532399	AM345	Fecal culture media	0hr	No sugar
SA532400	AM394	Fecal culture media	0hr	No sugar
SA532401	AM361	Fecal culture media	0hr	No sugar
SA532402	AM362	Fecal culture media	0hr	No sugar
SA532403	AM363	Fecal culture media	0hr	No sugar
SA532404	AM379	Fecal culture media	0hr	No sugar
SA532405	AM380	Fecal culture media	0hr	No sugar
SA532406	AM381	Fecal culture media	0hr	No sugar
SA532407	AM092	Fecal culture media	0hr	No sugar
SA532408	AM001	Fecal culture media	0hr	No sugar
SA532409	AM039	Fecal culture media	0hr	No sugar
SA532410	AM019	Fecal culture media	0hr	No sugar
SA532411	AM073	Fecal culture media	0hr	No sugar
SA532412	AM056	Fecal culture media	0hr	No sugar
SA532413	AM075	Fecal culture media	0hr	No sugar
SA532414	AM057	Fecal culture media	0hr	No sugar
SA532415	AM021	Fecal culture media	0hr	No sugar
SA532416	AM020	Fecal culture media	0hr	No sugar
SA532417	AM055	Fecal culture media	0hr	No sugar
SA532418	AM074	Fecal culture media	0hr	No sugar
SA532419	AM037	Fecal culture media	0hr	No sugar
SA532420	AM038	Fecal culture media	0hr	No sugar
SA532421	AM003	Fecal culture media	0hr	No sugar
SA532422	AM298	Fecal culture media	16hr	2'FL
SA532423	AM047	Fecal culture media	16hr	2'FL
SA532424	AM299	Fecal culture media	16hr	2'FL
SA532425	AM282	Fecal culture media	16hr	2'FL
SA532426	AM281	Fecal culture media	16hr	2'FL

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Collection:

Collection ID:	CO004647
Collection Summary:	Stool samples were collected from infants fed either 2'FL supplemented infant formula, standard infant formula, or human milk. Samples were then cultured anaerobically in the presence of prebiotics for 16hr and NMR metabolomics was performed.
Sample Type:	Feces
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR004663
Treatment Summary:	Stool samples were cultured anaerobically with 2’FL (FrieslandCampina Ingredients, The Netherlands) and/or purified GOS (FrieslandCampina Ingredients, The Netherlands) to assess their ability to utilize prebiotics and produce short chain fatty acids and dicarboxylic acids (responders or non-responders). The anaerobic culture supernatant was assessed by NMR metabolomics. Batch anaerobic fermentation was performed on 19 infant fecal samples at baseline, 1 month and 2 months of infant feeding. Samples were initially cultured anaerobically overnight at 37oC in Modified Reinforced Clostridial Media (ATCC 2107, per liter: 10g Tryptose, 10g Beef Extract, 3g Yeast Extract, 5g Dextrose, 5g NaCl, 1g soluble starch, 0.5g L-Cysteine HCl, 3g Sodium Acetate, 4mL 0.025% resazurin) as a rich starter culture medium (including carbon sources) and adjusted to an OD600 of 0.5 as previously described19. Each starter culture was then cultured anaerobically for 16 hours in the presence of 5g/L 2’FL, 5g/L GOS, or 2.5g/L 2’FL + 2.5g/L GOS. Aliquots of overnight starter cultures (1mL) were transferred into 15mL conical tubes containing 9mL of Fecal Collection Media, as described previously, containing per liter: 2 g peptone, 2 g yeast extract, 0.1 g NaCl, 0.04 g K2HPO4, 0.01 g MgSO4·7H2O, 0.01 g CaCl2·6H2O, 2 g NaHCO3, 0.005 g hemin (Sigma-Aldrich), 0.5 g l-cysteine HCl, 0.5 g bile salts, 2 mL Tween 80, 10 μL vitamin K and 4 mL of 0.025% (w/v) resazurin solution) using the designated prebiotic as the sole carbon (sugar) source19. Every time point of each subject was tested in triplicate, performing whole metagenome sequencing and metabolomics at baseline and again at 16 h for each sugar or control condition (no sugar, 5g/L 2’FL, 5g/L GOS, 5g/L dextrose, 2.5g/L 2’FL+ 2.5g/L GOS). After centrifugation at 2,000 x g for 2 minutes at 4oC, bacterial pellets were separated from supernatant and stored at -80oC for metabolomics analysis.

Treatment ID:

TR004663

Treatment Summary:

Stool samples were cultured anaerobically with 2’FL (FrieslandCampina Ingredients, The Netherlands) and/or purified GOS (FrieslandCampina Ingredients, The Netherlands) to assess their ability to utilize prebiotics and produce short chain fatty acids and dicarboxylic acids (responders or non-responders). The anaerobic culture supernatant was assessed by NMR metabolomics. Batch anaerobic fermentation was performed on 19 infant fecal samples at baseline, 1 month and 2 months of infant feeding. Samples were initially cultured anaerobically overnight at 37oC in Modified Reinforced Clostridial Media (ATCC 2107, per liter: 10g Tryptose, 10g Beef Extract, 3g Yeast Extract, 5g Dextrose, 5g NaCl, 1g soluble starch, 0.5g L-Cysteine HCl, 3g Sodium Acetate, 4mL 0.025% resazurin) as a rich starter culture medium (including carbon sources) and adjusted to an OD600 of 0.5 as previously described19. Each starter culture was then cultured anaerobically for 16 hours in the presence of 5g/L 2’FL, 5g/L GOS, or 2.5g/L 2’FL + 2.5g/L GOS. Aliquots of overnight starter cultures (1mL) were transferred into 15mL conical tubes containing 9mL of Fecal Collection Media, as described previously, containing per liter: 2 g peptone, 2 g yeast extract, 0.1 g NaCl, 0.04 g K2HPO4, 0.01 g MgSO4·7H2O, 0.01 g CaCl2·6H2O, 2 g NaHCO3, 0.005 g hemin (Sigma-Aldrich), 0.5 g l-cysteine HCl, 0.5 g bile salts, 2 mL Tween 80, 10 μL vitamin K and 4 mL of 0.025% (w/v) resazurin solution) using the designated prebiotic as the sole carbon (sugar) source19. Every time point of each subject was tested in triplicate, performing whole metagenome sequencing and metabolomics at baseline and again at 16 h for each sugar or control condition (no sugar, 5g/L 2’FL, 5g/L GOS, 5g/L dextrose, 2.5g/L 2’FL+ 2.5g/L GOS). After centrifugation at 2,000 x g for 2 minutes at 4oC, bacterial pellets were separated from supernatant and stored at -80oC for metabolomics analysis.

Sample Preparation:

Sampleprep ID:	SP004660
Sampleprep Summary:	All the test samples were thawed on ice on the day of the data collection. The samples were centrifuged at 10,000 x gn for 5 min at 4⁰C and 600 μL of supernatants were filtered at 12,000 x gn for 90-120 min at 4⁰C using pre-washed 3 kDa spin filters (VWR, 82031-344). The NMR buffer containing 100 mM phosphate buffer in D2O, pH 7.3, and 1.0 mM TMSP (3-Trimethylsilyl 2,2,3,3-d4 propionate) was added to 325-450uL of the filtrate. The final sample volume was 600 μL. The experiments were conducted using 550 μL samples in 103.5 mm x 5 mm NMR tubes (Wilmad Lab-Glass, WG-1000-4).

Sampleprep ID:

SP004660

Sampleprep Summary:

All the test samples were thawed on ice on the day of the data collection. The samples were centrifuged at 10,000 x gn for 5 min at 4⁰C and 600 μL of supernatants were filtered at 12,000 x gn for 90-120 min at 4⁰C using pre-washed 3 kDa spin filters (VWR, 82031-344). The NMR buffer containing 100 mM phosphate buffer in D2O, pH 7.3, and 1.0 mM TMSP (3-Trimethylsilyl 2,2,3,3-d4 propionate) was added to 325-450uL of the filtrate. The final sample volume was 600 μL. The experiments were conducted using 550 μL samples in 103.5 mm x 5 mm NMR tubes (Wilmad Lab-Glass, WG-1000-4).

Analysis:

Analysis ID:	AN007507
Laboratory Name:	Translational Metabolomics Facility (RRID: SCR_022636) - Cincinnati Children's Hospital Medical Center
Analysis Type:	NMR
Acquisition Date:	12/20/2021-01/06/2022
Operator Name:	Miki Watanabe-Chailland
Detector Type:	FT-NMR
Num Factors:	8
Num Metabolites:	35
Units:	AUC

NMR:

NMR ID:	NM000323
Analysis ID:	AN007507
Instrument Name:	Bruker Avance III
Instrument Type:	FT-NMR
NMR Experiment Type:	1D-1H
Field Frequency Lock:	Deuterium
Spectrometer Frequency:	600 MHz
NMR Probe:	Prodigy 5mm BBI
NMR Solvent:	D20
NMR Tube Size:	103.5 mm x 5 mm
Shimming Method:	Topshim
Pulse Sequence:	noesygppr1d