Evaluating the prebiotic activity of arabinogalactan on the human gut microbiota using 16S rRNA gene sequencing and Raman-activated cell sorting

Abstract

Background: Arabinogalactan is a complex plant-derived polysaccharide proposed to function as a selective prebiotic, yet the microbial taxa directly involved in its metabolism and the cooperative dynamics within the gut microbiota remain incompletely defined.

Methods: Here, we combined community-level sequencing with targeted single-cell activity profiling to investigate how arabinogalactan shapes gut microbial composition and function. Fecal samples from ten healthy individuals were incubated ex vivo with arabinogalactan, and microbial responses were assessed using 16S rRNA gene amplicon sequencing alongside Raman-activated cell sorting (RACS) and coculture experiments.

Results: Arabinogalactan consistently enriched Bifidobacterium and Gemmiger across donors, with Bifidobacterium also responding to galactose and Gemmiger and Blautia stimulated by arabinose, the two monosaccharide components of arabinogalactan. RACS enabled the selective isolation of metabolically active arabinogalactan responders, including Bifidobacterium longum (B. longum) and Faecalibacterium prausnitzii, along with other strains from the phyla Actinomycetota, Bacteroidota, and Bacillota. Notably, coculture experiments revealed that B. longum not only degraded arabinogalactan efficiently but also supported the growth of non-degrading species via metabolic cross-feeding. These cooperative interactions highlight B. longum as a keystone species in arabinogalactan utilization and suggest broader community-level benefits from its activity.

Conclusion: Together, our findings demonstrate arabinogalactan’s bifidogenic effect and its potential to promote functionally important microbes within the gut ecosystem. This study also highlights the utility of RACS for linking microbial identity to function, enabling the targeted recovery of active strains from complex communities.

Keywords

Arabinogalactan / Bifidobacterium longum / gut microbiota / prebiotics / Raman-activated cell sorting / microbial cross-feeding

References

[1] Belkaid Y.Role of the microbiota in immunity and inflammation.Cell2014;157:121-41 PMCID:PMC4056765

[2] David LA,Carmody RN.Diet rapidly and reproducibly alters the human gut microbiome.Nature2014;505:559-63 PMCID:PMC3957428

[3] Turnbaugh PJ,Yatsunenko T.A core gut microbiome in obese and lean twins.Nature2009;457:480-4 PMCID:PMC2677729

[4] Qin J, Li R, Raes J, et al; MetaHIT Consortium. A human gut microbial gene catalogue established by metagenomic sequencing. Nature. 2010;464:59-65. PMCID:PMC3779803

[5] Eckburg PB,Bernstein CN.Diversity of the human intestinal microbial flora.Science2005;308:1635-8 PMCID:PMC1395357

[6] Human Microbiome Project Consortium. Structure, function and diversity of the healthy human microbiome.Nature2012;486:207-14 PMCID:PMC3564958

[7] Slavin J.Fiber and prebiotics: mechanisms and health benefits.Nutrients2013;5:1417-35 PMCID:PMC3705355

[8] Gibson GR,Sanders ME.Expert consensus document: The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics.Nat Rev Gastroenterol Hepatol2017;14:491-502

[9] Davani-Davari D,Karimzadeh I.Prebiotics: definition, types, sources, mechanisms, and clinical applications.Foods2019;8:92 PMCID:PMC6463098

[10] Holscher HD.Dietary fiber and prebiotics and the gastrointestinal microbiota.Gut Microbes2017;8:172-84 PMCID:PMC5390821

[11] Dion C,Ripoll C.Does larch arabinogalactan enhance immune function? A review of mechanistic and clinical trials.Nutr Metab2016;13:28 PMCID:PMC4828828

[12] Uauy R.The assessment of dietary adequacy based on nutrient intake data is a complex issue. Foreword.Br J Nutr2009;101 Suppl 2:S1

[13] Gibson GR.Dietary modulation of the human colonic microbiota: introducing the concept of prebiotics.J Nutr1995;125:1401-12

[14] Wang Y.Arabinogalactan utilization by Bifidobacterium longum subsp. longum NCC 2705 and Bacteroides caccae ATCC 43185 in monoculture and coculture.Microorganisms2020;8:1703 PMCID:PMC7693162

[15] Kim K.Rheological properties of arabinogalactan solutions related to the carbohydrate composition of different legumes.Korean J Food Preserv2023;30:785-96

[16] Rakhmanberdyeva RK,Senchenkova SN,Bobakulov KM.Structure of arabinogalactan and pectin from the Silybum marianum.Carbohydr Res2019;485:107797

[17] Wang M,He X,Huang Q.In vitro colonic fermentation of dietary fibers: fermentation rate, short-chain fatty acid production and changes in microbiota.Trends Food Sci Technol2019;88:1-9

[18] Sun Y,Zhang S.Prebiotic characteristics of arabinogalactans during in vitro fermentation through multi-omics analysis.Food Chem Toxicol2021;156:112522

[19] Victoria Obayomi O, Folakemi Olaniran A, Olugbemiga Owa S. Unveiling the role of functional foods with emphasis on prebiotics and probiotics in human health: a review.J Funct Foods2024;119:106337

[20] Wang Y,Ivusic Polic I,Lapointe G.Modulation of human gut microbiota composition and metabolites by arabinogalactan and Bifidobacterium longum subsp. longum BB536 in the Simulator of the Human Intestinal Microbial Ecosystem (SHIME®).J Funct Foods2021;87:104820

[21] Sasaki Y,Odamaki T.Novel 3-O-α-D-Galactosyl-α-L-arabinofuranosidase for the assimilation of gum arabic arabinogalactan protein in Bifidobacterium longum subsp. longum.Appl Environ Microbiol2021;87:e02690-20 PMCID:PMC8117759

[22] Onumpai C,Bonnin E.Microbial utilization and selectivity of pectin fractions with various structures.Appl Environ Microbiol2011;77:5747-54 PMCID:PMC3165253

[23] Lindstad LJ,Leivers S.Human gut faecalibacterium prausnitzii deploys a highly efficient conserved system to cross-feed on β-mannan-derived oligosaccharides.mBio2021;12:e0362820 PMCID:PMC8262883

[24] Culp EJ.Cross-feeding in the gut microbiome: ecology and mechanisms.Cell Host Microbe2023;31:485-99 PMCID:PMC10125260

[25] Cartmell A,Briggs JA.A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation.Nat Microbiol2018;3:1314-26 PMCID:PMC6217937

[26] Wang S,Yu C.Microbial collaborations and conflicts: unraveling interactions in the gut ecosystem.Gut Microbes2024;16:2296603 PMCID:PMC10761165

[27] Lombard V,Drula E,Henrissat B.The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res2014;42:D490-5 PMCID:PMC3965031

[28] Shi E,Wang X.Polysaccharides affect the utilization of β-carotene through gut microbiota investigated by in vitro and in vivo experiments.Food Res Int2023;174:113592

[29] Holmes ZC,Durand HK.Microbiota responses to different prebiotics are conserved within individuals and associated with habitual fiber intake.Microbiome2022;10:114 PMCID:PMC9336045

[30] Falony G,Vieira-Silva S.Population-level analysis of gut microbiome variation.Science2016;352:560-4

[31] Wang Q,Boonpawa R,Beekmann K.Use of physiologically based kinetic modeling to predict rat gut microbial metabolism of the isoflavone daidzein to S-equol and its consequences for ERα activation.Mol Nutr Food Res2020;64:e1900912 PMCID:PMC7154660

[32] Bénard MV,Wortelboer K.Anaerobic feces processing for fecal microbiota transplantation improves viability of obligate anaerobes.Microorganisms2023;11:2238 PMCID:PMC10535047

[33] Salonen A,Jalanka-Tuovinen J.Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: effective recovery of bacterial and archaeal DNA using mechanical cell lysis.J Microbiol Methods2010;81:127-34

[34] Pjevac P,Schwarz J.An economical and flexible dual barcoding, two-step PCR approach for highly multiplexed amplicon sequencing.Front Microbiol2021;12:669776 PMCID:PMC8173057

[35] Berry D,Lee TK.Tracking heavy water (D₂O) incorporation for identifying and sorting active microbial cells.Proc Natl Acad Sci U S A2015;112:E194-203 PMCID:PMC4299247

[36] Eichorst SA,Woyke T,Wagner M.Advancements in the application of NanoSIMS and Raman microspectroscopy to investigate the activity of microbial cells in soils.FEMS Microbiol Ecol2015;91:fiv106 PMCID:PMC4629873

[37] Cui D,Wang Y,Zhang C.In situ identification of environmental microorganisms with Raman spectroscopy.Environ Sci Ecotechnol2022;11:100187 PMCID:PMC9488013

[38] Riva A,Cruz-Rubio JM.Identification of inulin-responsive bacteria in the gut microbiota via multi-modal activity-based sorting.Nat Commun2023;14:8210 PMCID:PMC10721620

[39] Henry DG,Gillmore G,Emmings JF.Assessing low-maturity organic matter in shales using Raman spectroscopy: effects of sample preparation and operating procedure.Int J Coal Geol2018;191:135-51

[40] Caro TA,Brown G,Kopf SH.Single-cell measurement of microbial growth rate with Raman microspectroscopy.FEMS Microbiol Ecol2024;100:fiae110 PMCID:PMC11347945

[41] Stöckel S,Neugebauer U,Popp J.The application of Raman spectroscopy for the detection and identification of microorganisms.J Raman Spectrosc2016;47:89-109

[42] Lee KS,Palatinszky M.Optofluidic Raman-activated cell sorting for targeted genome retrieval or cultivation of microbial cells with specific functions.Nat Protoc2021;16:634-76

[43] Lee KS,Pereira FC.An automated Raman-based platform for the sorting of live cells by functional properties.Nat Microbiol2019;4:1035-48

[44] Wright ES,Noguera DR.DECIPHER, a search-based approach to chimera identification for 16S rRNA sequences.Appl Environ Microbiol2012;78:717-25 PMCID:PMC3264099

[45] Untergasser A,Rao X,Geurts R.Primer3Plus, an enhanced web interface to Primer3.Nucleic Acids Res2007;35:W71-4 PMCID:PMC1933133

[46] R Studio Team. A language and environment for statistical computing. Available from: https://www.R-project.org. [Last accessed on 6 Aug 2025]

[47] Valero-Mora PM.ggplot2: elegant graphics for data analysis.J Stat Softw2010;

[48] Oksanen J,Blanchet FG. Package ‘vegan’. Available from: https://cran.r-project.org/web/packages/vegan/vegan.pdf. [Last accessed on 6 Aug 2025]

[49] Benjamini Y.Controlling the false discovery rate: a practical and powerful approach to multiple testing.J R Stat Soc Ser B Stat Methodol1995;57:289-300

[50] Love MI,Anders S.Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.Genome Biol2014;15:550 PMCID:PMC4302049

[51] Letunic I.Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool.Nucleic Acids Res2024;52:W78-82 PMCID:PMC11223838

[52] Kau AL,Griffin NW,Gordon JI.Human nutrition, the gut microbiome and the immune system.Nature2011;474:327-36 PMCID:PMC3298082

[53] Valdes AM,Segal E.Role of the gut microbiota in nutrition and health.BMJ2018;361:k2179 PMCID:PMC6000740

[54] O’Callaghan A.Bifidobacteria and their role as members of the human gut microbiota.Front Microbiol2016;7:925 PMCID:PMC4908950

[55] Rinninella E,Cintoni M.What is the healthy gut microbiota composition? A changing ecosystem across age, environment, diet, and diseases.Microorganisms2019;7:14 PMCID:PMC6351938

[56] Hill C,Reid G.Expert consensus document. The International Scientific Association for Probiotics and Prebiotics consensus statement on the scope and appropriate use of the term probiotic.Nat Rev Gastroenterol Hepatol2014;11:506-14

[57] Hughes RL,Swanson KS.The prebiotic potential of inulin-type fructans: a systematic review.Adv Nutr2022;13:492-529 PMCID:PMC8970830

[58] Scott KP,Sheridan PO,Duncan SH.The influence of diet on the gut microbiota.Pharmacol Res2013;69:52-60

[59] Bindels LB,Cani PD.Towards a more comprehensive concept for prebiotics.Nat Rev Gastroenterol Hepatol2015;12:303-10

[60] Xiao M,Duan H.Cross-feeding of bifidobacteria promotes intestinal homeostasis: a lifelong perspective on the host health.NPJ Biofilms Microbiomes2024;10:47 PMCID:PMC11186840

[61] González R,Malinen E,Vaughan EE.Differential transcriptional response of Bifidobacterium longum to human milk, formula milk, and galactooligosaccharide.Appl Environ Microbiol2008;74:4686-94 PMCID:PMC2519361

[62] Liu M,Zhang N.Preparation of polysaccharides from Crepis tectorum Linn. and the regulation effects on intestinal microbiota.Process Biochem2023;130:50-66

[63] Gao X,Li T.CAZymes-associated method to explore glycans that mitigate DSS-induced colitis via targeting Bacteroides cellulosilyticus.Int J Biol Macromol2024;258:128694

[64] Peterson CT,Iablokov SN.16S rRNA gene profiling and genome reconstruction reveal community metabolic interactions and prebiotic potential of medicinal herbs used in neurodegenerative disease and as nootropics.PLoS One2019;14:e0213869 PMCID:PMC6424447

[65] Aguirre M,Venema K.The gut microbiota from lean and obese subjects contribute differently to the fermentation of arabinogalactan and inulin.PLoS One2016;11:e0159236 PMCID:PMC4943740

[66] He X,Li Y.Faecalibacterium prausnitzii: a next-generation probiotic in gut disease improvement.Can J Infect Dis Med Microbiol2021;2021:1-10

[67] Fujita K,Kitahara K.Degradation of plant arabinogalactan proteins by intestinal bacteria: characteristics and functions of the enzymes involved.Appl Microbiol Biotechnol2019;103:7451-7

[68] Hirmas B,Orellana G.Metabolic modeling and bidirectional culturing of two gut microbes reveal cross-feeding interactions and protective effects on intestinal cells.mSystems2022;7:e0064622 PMCID:PMC9600892

[69] Steinert RE,Sadabad MS.Microbial micronutrient sharing, gut redox balance and keystone taxa as a basis for a new perspective to solutions targeting health from the gut.Gut Microbes2025;17:2477816 PMCID:PMC11913388

[70] Ku S,Jang MJ.The role of Bifidobacterium in longevity and the future of probiotics.Food Sci Biotechnol2024;33:2097-110 PMCID:PMC11315853

[71] Wong CB,Xiao J.Beneficial effects of Bifidobacterium longum subsp. longum BB536 on human health: modulation of gut microbiome as the principal action.J Funct Foods2019;54:506-19

[72] Gossling J.Gemmiger formicilis, n.gen., n.sp., an anaerobic budding bacterium from intestines.Int J Syst Bacteriol1975;25:202-7

[73] Salanitro JP,Goodman JR.Morphological and physiological characteristics of Gemmiger formicilis isolated from chicken ceca.Appl Environ Microbiol1976;32:623-32 PMCID:PMC170317

[74] Zenner C,Riedel T.Early-life immune system maturation in chickens using a synthetic community of cultured gut bacteria.mSystems2021;6:e01300-20 PMCID:PMC8269260

[75] Fitzgerald CB,Sutton TDS.Comparative analysis of Faecalibacterium prausnitzii genomes shows a high level of genome plasticity and warrants separation into new species-level taxa.BMC Genomics2018;19:931 PMCID:PMC6295017

[76] Guo W,Yue H.Associations of intermittent hypoxia burden with gut microbiota dysbiosis in adult patients with obstructive sleep apnea.Nat Sci Sleep2024;16:1483-95 PMCID:PMC11438448

[77] Qian L,Qin H.Probiotics and dietary intervention modulate the colonic mucosa-associated microbiota in high-fat diet populations.Turk J Gastroenterol2020;31:295-304 PMCID:PMC7236651

[78] Arnold JW,Fabela S.The pleiotropic effects of prebiotic galacto-oligosaccharides on the aging gut.Microbiome2021;9:31 PMCID:PMC7845053

[79] Alberto F,Sulzenbacher G,Czjzek M.The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases.J Biol Chem2004;279:18903-10

[80] Andersen JM,Abou Hachem M.Transcriptional analysis of oligosaccharide utilization by Bifidobacterium lactis Bl-04.BMC Genomics2013;14:312 PMCID:PMC3684542

[81] Fujita K,Lixia P.Bifidobacterial GH146 β-L-arabinofuranosidase for the removal of β1,3-L-arabinofuranosides on plant glycans.Appl Microbiol Biotechnol2024;108:199 PMCID:PMC10850190

[82] Fujita K,Kaneko S,Tsumuraya Y.Degradative enzymes for type II arabinogalactan side chains in Bifidobacterium longum subsp. longum.Appl Microbiol Biotechnol2019;103:1299-310

[83] Fujita K,Obuchi E,Suganuma T.Characterization of a novel β-L-arabinofuranosidase in Bifidobacterium longum: functional elucidation of a DUF1680 protein family member.J Biol Chem2014;289:5240-9 PMCID:PMC3931080

[84] Xu J,Jia M,Zhu W.Metatranscriptomic analysis of colonic microbiota’s functional response to different dietary fibers in growing pigs.Anim Microbiome2021;3:45 PMCID:PMC8254964

[85] Li W,Liang H.Genomic and functional diversity of the human-derived isolates of Faecalibacterium.Front Microbiol2024;15:1379500 PMCID:PMC11169845

[86] Barlow JT,Ismagilov RF.A quantitative sequencing framework for absolute abundance measurements of mucosal and lumenal microbial communities.Nat Commun2020;11:2590 PMCID:PMC7244552