Skip to main content
SpringerLink
Log in

Protocols for the Use of Gut Models to Study the Potential Contribution of the Gut Microbiota to Human Nutrition Through the Production of Short-Chain Fatty Acids

  • Protocol
  • First Online:
Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

  • 731 Accesses

Abstract

The colonic microbiota influences human energy status through the metabolic activity of the taxonomically diverse prokaryotic residents that number up to 1012 cells per gram. The principal means by which this happens is probably via short-chain fatty acids (SCFAs) (mainly acetate, propionate and butyrate), which are continually produced by fermentation of dietary fibre, absorbed through the colonic epithelium, transported via the hepatic portal vein to the liver and then converted to glucose and other lipids. SCFAs may also regulate appetite via G protein-coupled receptor (GPR43) activation and signalling. Since the colonic microbiota is inaccessible for routine investigation, microbiologists have used human faeces as a surrogate for intestinal contents, animal models and various in vitro systems. These range in complexity from batch cultures of isolated gut bacteria through defined consortia grown in batch and continuous culture to multistage continuous culture models that reproduce features of the proximal and distal colons. Such systems have been used to investigate the metabolism of gut bacteria for several decades and can be adopted for studies specifically focussing on SCFA production in the context of nutrition/obesity. SCFAs generated by bacterial fermentation can be analysed using gas chromatography, or more inclusive data can be obtained via metabonomics/metabolomics. Whilst culture and FISH provide a useful means of bacteriological analysis, next-generation sequencing (NGS) has facilitated major advances in our understanding of this complex ecosystem. The following protocol details the establishment of a three-stage continuous culture microcosm of the human colon and outlines options for bacteriological and metabolite analyses.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Leser TD, Molbak L (2009) Better living through microbial action: the benefits of the mammalian gastrointestinal microbiota on the host. Environ Microbiol 11(9):2194–2206. doi:10.1111/j.1462-2920.2009.01941.x

    Article  CAS  PubMed  Google Scholar 

  2. Kau AL, Ahern PP, Griffin NW, Goodman AL, Gordon JI (2011) Human nutrition, the gut microbiome and the immune system. Nature 474:327–336. doi:10.1038/nature10213

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Stappenbeck TS, Hooper LV, Gordon JI (2002) Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells. Proc Natl Acad Sci U S A 99:15451–15455. doi:10.1073/pnas.202604299202604299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Macfarlane GT, Englyst HN (1986) Starch utilization by the human large intestinal microflora. J Appl Bacteriol 60(3):195–201

    Article  CAS  PubMed  Google Scholar 

  5. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Guarner F, Pedersen O, de Vos WM, Brunak S, Dore J, Meta HITC, Antolin M, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Merieux A, Melo Minardi R, M’Rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P (2011) Enterotypes of the human gut microbiome. Nature 473(7346):174–180. doi:10.1038/nature09944

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Ley RE, Backhed F, Turnbaugh P, Lozupone CA, Knight RD, Gordon JI (2005) Obesity alters gut microbial ecology. Proc Natl Acad Sci U S A 102(31):11070–11075. doi:10.1073/pnas.0504978102

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI (2006) An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444(7122):1027–1031. doi:10.1038/nature05414

    Article  PubMed  Google Scholar 

  8. Macfarlane GT, Cummings JH, Allison C (1986) Protein degradation by human intestinal bacteria. J Gen Microbiol 132(6):1647–1656

    CAS  PubMed  Google Scholar 

  9. Ridaura VK, Faith JJ, Rey FE, Cheng J, Duncan AE, Kau AL, Griffin NW, Lombard V, Henrissat B, Bain JR, Muehlbauer MJ, Ilkayeva O, Semenkovich CF, Funai K, Hayashi DK, Lyle BJ, Martini MC, Ursell LK, Clemente JC, Van Treuren W, Walters WA, Knight R, Newgard CB, Heath AC, Gordon JI (2013) Gut microbiota from twins discordant for obesity modulate metabolism in mice. Science 341(6150):1241214. doi:10.1126/science.1241214

    Article  PubMed  Google Scholar 

  10. Turnbaugh PJ, Hamady M, Yatsunenko T, Cantarel BL, Duncan A, Ley RE, Sogin ML, Jones WJ, Roe BA, Affourtit JP, Egholm M, Henrissat B, Heath AC, Knight R, Gordon JI (2009) A core gut microbiome in obese and lean twins. Nature 457(7228):480–484. doi:10.1038/nature07540

    Article  CAS  PubMed  Google Scholar 

  11. Kalliomäki M, Collado MC, Salminen S, Isolauri E (2008) Early differences in fecal microbiota composition in children may predict overweight. Am J Clin Nutr 87(3):534–538

    PubMed  Google Scholar 

  12. Bergman EN (1990) Energy contributions of volatile fatty acids from the gastrointestinal tract in various species. Physiol Rev 70(2):567–590

    CAS  PubMed  Google Scholar 

  13. Bloemen JG, Venema K, van de Poll MC, Olde Damink SW, Buurman WA, Dejong CH (2009) Short chain fatty acids exchange across the gut and liver in humans measured at surgery. Clin Nutr 28(6):657–661. doi:10.1016/j.clnu.2009.05.011

    Article  CAS  PubMed  Google Scholar 

  14. Le Poul E, Loison C, Struyf S, Springael JY, Lannoy V, Decobecq ME, Brezillon S, Dupriez V, Vassart G, Van Damme J, Parmentier M, Detheux M (2003) Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. J Biol Chem 278(28):25481–25489. doi:10.1074/jbc.M301403200

    Article  PubMed  Google Scholar 

  15. Engelstoft MS, Egerod KL, Holst B, Schwartz TW (2008) A gut feeling for obesity: 7TM sensors on enteroendocrine cells. Cell Metab 8(6):447–449. doi:10.1016/j.cmet.2008.11.004

    Article  CAS  PubMed  Google Scholar 

  16. Chambers ES, Viardot A, Psichas A, Morrison DJ, Murphy KG, Zac-Varghese SE, MacDougall K, Preston T, Tedford C, Finlayson GS, Blundell JE, Bell JD, Thomas EL, Mt-Isa S, Ashby D, Gibson GR, Kolida S, Dhillo WS, Bloom SR, Morley W, Clegg S, Frost G (2014) Effects of targeted delivery of propionate to the human colon on appetite regulation, body weight maintenance and adiposity in overweight adults. Gut. doi:10.1136/gutjnl-2014-307913

    PubMed  PubMed Central  Google Scholar 

  17. Frost G, Sleeth ML, Sahuri-Arisoylu M, Lizarbe B, Cerdan S, Brody L, Anastasovska J, Ghourab S, Hankir M, Zhang S, Carling D, Swann JR, Gibson G, Viardot A, Morrison D, Louise Thomas E, Bell JD (2014) The short-chain fatty acid acetate reduces appetite via a central homeostatic mechanism. Nat Commun 5:3611. doi:10.1038/ncomms4611

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Degnan BA, Macfarlane S, Quigley ME, Macfarlane GT (1997) Starch utilization by Bacteroides ovatus isolated from the human large intestine. Curr Microbiol 34(5):290–296

    Article  CAS  PubMed  Google Scholar 

  19. Gibson GR, Cummings JH, Macfarlane GT (1988) Competition for hydrogen between sulphate-reducing bacteria and methanogenic bacteria from the human large intestine. J Appl Bacteriol 65(3):241–247

    Article  CAS  PubMed  Google Scholar 

  20. Macfarlane S, McBain AJ, Macfarlane GT (1997) Consequences of biofilm and sessile growth in the large intestine. Adv Dent Res 11(1):59–68

    Article  CAS  PubMed  Google Scholar 

  21. Macfarlane S, Woodmansey EJ, Macfarlane GT (2005) Colonization of mucin by human intestinal bacteria and establishment of biofilm communities in a two-stage continuous culture system. Appl Environ Microbiol 71(11):7483–7492. doi:10.1128/AEM.71.11.7483-7492.2005

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. McBain AJ, Macfarlane GT (2001) Modulation of genotoxic enzyme activities by non-digestible oligosaccharide metabolism in in-vitro human gut bacterial ecosystems. J Med Microbiol 50(9):833–842

    Article  CAS  PubMed  Google Scholar 

  23. McBain AJ (2009) Chapter 4: in vitro biofilm models: an overview. Adv Appl Microbiol 69:99–132

    Article  CAS  PubMed  Google Scholar 

  24. Macfarlane GT, Macfarlane S (2007) Models for intestinal fermentation: association between food components, delivery systems, bioavailability and functional interactions in the gut. Curr Opin Biotechnol 18(2):156–162. doi:10.1016/j.copbio.2007.01.011

    Article  CAS  PubMed  Google Scholar 

  25. Gibson GR, Cummings JH, Macfarlane GT (1988) Use of a three-stage continuous culture system to study the effect of mucin on dissimilatory sulfate reduction and methanogenesis by mixed populations of human gut bacteria. Appl Environ Microbiol 54(11):2750–2755

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Allison C, McFarlan C, Macfarlane GT (1989) Studies on mixed populations of human intestinal bacteria grown in single-stage and multistage continuous culture systems. Appl Environ Microbiol 55(3):672–678

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Macfarlane GT, Gibson GR (1991) Co-utilization of polymerized carbon sources by Bacteroides ovatus grown in a two-stage continuous culture system. Appl Environ Microbiol 57(1):1–6

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Macfarlane GT, Gibson GR, Cummings JH (1992) Comparison of fermentation reactions in different regions of the human colon. J Appl Bacteriol 72(1):57–64

    CAS  PubMed  Google Scholar 

  29. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, Fierer N, Knight R (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108(Suppl 1):4516–4522. doi:10.1073/pnas.1000080107

    Article  CAS  PubMed  Google Scholar 

  30. Huda-Faujan N, Abdulamir AS, Fatimah AB, Anas OM, Shuhaimi M, Yazid AM, Loong YY (2010) The impact of the level of the intestinal short chain Fatty acids in inflammatory bowel disease patients versus healthy subjects. Open Biochem J 4:53–58

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Furusawa Y, Obata Y, Fukuda S, Endo TA, Nakato G, Takahashi D, Nakanishi Y, Uetake C, Kato K, Kato T, Takahashi M, Fukuda NN, Murakami S, Miyauchi E, Hino S, Atarashi K, Onawa S, Fujimura Y, Lockett T, Clarke JM, Topping DL, Tomita M, Hori S, Ohara O, Morita T, Koseki H, Kikuchi J, Honda K, Hase K, Ohno H (2013) Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells. Nature 504(7480):446–450. doi:10.1038/nature12721

    Article  CAS  PubMed  Google Scholar 

  32. Bourriaud C, Robins RJ, Martin L, Kozlowski F, Tenailleau E, Cherbut C, Michel C (2005) Lactate is mainly fermented to butyrate by human intestinal microfloras but inter-individual variation is evident. J Appl Microbiol 99(1):201–212. doi:10.1111/j.1365-2672.2005.02605.x

    Article  CAS  PubMed  Google Scholar 

  33. Zheng X, Qiu Y, Zhong W, Baxter S, Su M, Li Q, Xie G, Ore BM, Qiao S, Spencer MD, Zeisel SH, Zhou Z, Zhao A, Jia W (2013) A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids. Metabolomics 9(4):818–827. doi:10.1007/s11306-013-0500-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgement

AJM gratefully acknowledges Professor George Tennant Macfarlane (1958–2015) for permission to use his designs and diagrams in this article, for inspiration and insight and for his significant contributions to intestinal microbiology over more than 30 years.

Author information

Authors and Affiliations

  1. Manchester Pharmacy School, The University of Manchester, Oxford Road, Manchester, M13 9PT, UK

    Andrew J McBain, Ruth Ledder & Gavin Humphreys

Authors
  1. Andrew J McBain
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ruth Ledder
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Gavin Humphreys
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Andrew J McBain .

Editor information

Editors and Affiliations

  1. School of Biological Sciences, University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer-Verlag Berlin Heidelberg

About this protocol

Cite this protocol

McBain, A.J., Ledder, R., Humphreys, G. (2015). Protocols for the Use of Gut Models to Study the Potential Contribution of the Gut Microbiota to Human Nutrition Through the Production of Short-Chain Fatty Acids. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_127

Download citation

  • DOI: https://doi.org/10.1007/8623_2015_127

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-53113-6

  • Online ISBN: 978-3-662-53115-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation