Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol Extension
  • Published:

An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality

Abstract

Knowledge of the effect of foods on gut microbiota composition and functionality is expanding. To isolate the effect of single foods and/or single nutrients (i.e., fiber, polyphenols), this protocol describes an in vitro batch fermentation procedure to be carried out after an in vitro gastrointestinal digestion. Therefore, this is an extension of the previous protocol described by Brodkorb et al. (2019) for studying in vitro digestion. The current protocol uses an oligotrophic fermentation medium with peptone and a high concentration of fecal inoculum from human fecal samples both to provide the microbiota and as the main source of nutrients for the bacteria. This protocol is recommended for screening work to be performed when many food samples are to be studied. It has been used successfully to study gut microbiota fermentation of different foodstuffs, giving insights into their functionality, community structure or ability to degrade particular substances, which can contribute to the development of personalized nutrition strategies. The procedure does not require a specific level of expertise. The protocol takes 4–6 h for preparation of fermentation tubes and 20 h for incubation.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: In vitro fermentation process.
Fig. 2: Reproducibility assessment.
Fig. 3: Microbial functionality affected by specific health conditions.
Fig. 4: Differences in gut microbial community structure after fermenting different foods.

Similar content being viewed by others

Data availability

The data shown in Fig. 4 are available from the supporting primary research paper previously published by Pérez-Burillo et al.17. The data presented in Figs. 2 and 3 were generated for this protocol. Source data are provided with this paper.

References

  1. Flint, H. J., Duncan, S. H., Scott, K. P. & Louis, P. Links between diet, gut microbiota composition and gut metabolism. Proc. Nutr. Soc. 74, 13–22 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Salazar, N. et al. Exopolysaccharides produced by Bifidobacterium longum IPLA E44 and Bifidobacterium animalis subsp. lactis IPLA R1 modify the composition and metabolic activity of human faecal microbiota in pH-controlled batch cultures. Int. J. Food Microbiol. 135, 260–267 (2009).

    Article  CAS  PubMed  Google Scholar 

  3. Power, S. E., O’Toole, P. W., Stanton, C., Ross, R. P. & Fitzgerald, G. F. Intestinal microbiota, diet and health. Br. J. Nutr. 111, 387–402 (2014).

    Article  CAS  PubMed  Google Scholar 

  4. Shankar, V. et al. Differences in gut metabolites and microbial composition and functions between Egyptian and U.S. children are consistent with their diets. mSystems 2, e00169–16 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  5. David, L. A. et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  6. De Filippo, C. et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl Acad. Sci. USA 107, 14691–14696 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  7. Venema, K. & van den Abbeele, P. Experimental models of the gut microbiome. Best Pract. Res. Clin. Gastroenterol. 27, 115–126 (2013).

    Article  CAS  PubMed  Google Scholar 

  8. Molly, K., Vande Woestyne, M. & Verstraete, W. Development of a 5-step multi-chamber reactor as a simulation of the human intestinal microbial ecosystem. Appl. Microbiol. Biotechnol. 39, 254–258 (1993).

    Article  CAS  PubMed  Google Scholar 

  9. Agans, R. et al. Dietary fatty acids sustain the growth of the human gut microbiota. Appl. Environ. Microbiol. 84, e01525–18 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Wiese, M. et al. CoMiniGut—a small volume in vitro colon model for the screening of gut microbial fermentation processes. PeerJ 6, e4268 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Ludwig, I. A., Paz de Peña, M., Concepción, C. & Alan, C. Catabolism of coffee chlorogenic acids by human colonic microbiota: colonic catabolism of coffee chlorogenic acids. BioFactors 39, 623–632 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Coles, L. T., Moughan, P. J. & Darragh, A. J. In vitro digestion and fermentation methods, including gas production techniques, as applied to nutritive evaluation of foods in the hindgut of humans and other simple-stomached animals. Anim. Feed Sci. Technol. 123–124, 421–444 (2005).

    Article  Google Scholar 

  13. Wang, M. et al. In vitro colonic fermentation of dietary fibers: fermentation rate, short-chain fatty acid production and changes in microbiota. Trends Food Sci. Technol. 88, 1–9 (2019).

    Article  CAS  Google Scholar 

  14. Mould, F. L., Morgan, R., Kliem, K. E. & Krystallidou, E. A review and simplification of the in vitro incubation medium. Anim. Feed Sci. Technol. 123–124, 155–172 (2005).

    Article  Google Scholar 

  15. Brodkorb, A. et al. INFOGEST static in vitro simulation of gastrointestinal food digestion. Nat. Protoc. 14, 991–1014 (2019).

    Article  CAS  PubMed  Google Scholar 

  16. Pérez-Burillo, S., Rufián-Henares, J. A. & Pastoriza, S. Towards an improved global antioxidant response method (GAR+): physiological-resembling in vitro digestion-fermentation method. Food Chem 239, 1253–1262 (2018).

    Article  PubMed  Google Scholar 

  17. Pérez-Burillo, S. et al. Effect of food thermal processing on the composition of the gut microbiota. J. Agric. Food Chem. 66, 11500–11509 (2018).

    Article  PubMed  Google Scholar 

  18. Pérez-Burillo, S. et al. Effect of in vitro digestion-fermentation on green and roasted coffee bioactivity: the role of the gut microbiota. Food Chem. 279, 252–259 (2019).

    Article  PubMed  Google Scholar 

  19. Pérez-Burillo, S. et al. Potential probiotic salami with dietary fiber modulates antioxidant capacity, short chain fatty acid production and gut microbiota community structure. LWT 105, 355–362 (2019).

    Article  Google Scholar 

  20. Pérez-Burillo, S. et al. Spent coffee grounds extract, rich in mannooligosaccharides, promotes a healthier gut microbial community in a dose-dependent manner. J. Agric. Food Chem. 67, 2500–2509 (2019).

    Article  PubMed  Google Scholar 

  21. Pérez-Burillo, S., Rajakaruna, S., Pastoriza, S., Paliy, O. & Rufián-Henares, J. A. Bioactivity of food melanoidins is mediated by gut microbiota. Food Chem. 316, 126309 (2020).

    Article  PubMed  Google Scholar 

  22. Rocchetti, G. et al. Transformation of polyphenols found in pigmented gluten-free flours during in vitro large intestinal fermentation. Food Chem. 298, 125068 (2019).

    Article  CAS  PubMed  Google Scholar 

  23. Jin, J. B. et al. Supplementation with Chlorella vulgaris, Chlorella protothecoides, and Schizochytrium sp. increases propionate-producing bacteria in in vitro human gut fermentation. J. Sci. Food Agric. 100, 2938–2945 (2020).

    Article  CAS  PubMed  Google Scholar 

  24. Selma, M. V., Espín, J. C. & Tomás-Barberán, F. A. Interaction between phenolics and gut microbiota: role in human health. J. Agric. Food Chem. 57, 6485–6501 (2009).

    Article  CAS  PubMed  Google Scholar 

  25. Rowland, I. et al. Gut microbiota functions: metabolism of nutrients and other food components. Eur. J. Nutr. 57, 1–24 (2018).

    Article  CAS  PubMed  Google Scholar 

  26. Tomas‐Barberan, F. et al. In vitro transformation of chlorogenic acid by human gut microbiota. Mol. Nutr. Food Res. 58, 1122–1131 (2014).

    Article  PubMed  Google Scholar 

  27. Saura‐Calixto, F. et al. Proanthocyanidin metabolites associated with dietary fibre from in vitro colonic fermentation and proanthocyanidin metabolites in human plasma. Mol. Nutr. Food Res. 54, 939–946 (2010).

    Article  PubMed  Google Scholar 

  28. Roowi, S. et al. Green tea flavan-3-ols: colonic degradation and urinary excretion of catabolites by humans. J. Agric. Food Chem. 58, 1296–1304 (2010).

    Article  CAS  PubMed  Google Scholar 

  29. Jaganath, I. B., Mullen, W., Lean, M. E. J., Edwards, C. A. & Crozier, A. In vitro catabolism of rutin by human fecal bacteria and the antioxidant capacity of its catabolites. Free Radic. Biol. Med. 47, 1180–1189 (2009).

    Article  CAS  PubMed  Google Scholar 

  30. Serra, A. et al. Metabolic pathways of the colonic metabolism of flavonoids (flavonols, flavones and flavanones) and phenolic acids. Food Chem. 130, 383–393 (2012).

    Article  CAS  Google Scholar 

  31. Pinta, M. N. et al. In vitro gut metabolism of [U-13C]-quinic acid, the other hydrolysis product of chlorogenic acid. Mol. Nutr. Food Res. 62, 1800396 (2018).

  32. Hidalgo, M. et al. Metabolism of anthocyanins by human gut microflora and their influence on gut bacterial growth. J. Agric. Food Chem. 60, 3882–3890 (2012).

    Article  CAS  PubMed  Google Scholar 

  33. Marín, L., Miguélez, E. M., Villar, C. J. & Lombó, F. Bioavailability of dietary polyphenols and gut microbiota metabolism: antimicrobial properties. Biomed. Res. Int. https://doi.org/10.1155/2015/905215 (2015).

  34. Stevens, J. F. & Maier, C. S. The chemistry of gut microbial metabolism of polyphenols. Phytochem. Rev. 15, 425–444 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Fuertes, Á. et al. Adaptation of the human gut microbiota metabolic network during the first year after birth. Front. Microbiol. 10, 848 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Ríos-Covián, D. et al. Intestinal short chain fatty acids and their link with diet and human health. Front. Microbiol. 7, 185 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Shen, Q., Chen, Y. A. & Tuohy, K. M. A comparative in vitro investigation into the effects of cooked meats on the human faecal microbiota. Anaerobe 16, 572–577 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Poelaert, C. et al. Cooking has variable effects on the fermentability in the large intestine of the fraction of meats, grain legumes, and insects that is resistant to digestion in the small intestine in an in vitro model of the pig’s gastrointestinal tract. J. Agric. Food Chem. 65, 435–444 (2017).

    Article  CAS  PubMed  Google Scholar 

  39. Lee, D. K. et al. The combination of mixed lactic acid bacteria and dietary fiber lowers serum cholesterol levels and fecal harmful enzyme activities in rats. Arch. Pharm. Res. 34, 23–29 (2011).

    Article  CAS  PubMed  Google Scholar 

  40. Molska, M. & Regula, J. Potential mechanisms of probiotics action in the prevention and treatment of colorectal cancer. Nutrients 11, 2453 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  41. Pham, V. T. & Mohajeri, M. H. The application of in vitro human intestinal models on the screening and development of pre- and probiotics. Benef. Microbes 9, 725–742 (2018).

    Article  CAS  PubMed  Google Scholar 

  42. Gu, F. et al. In vitro fermentation behavior of isomalto/malto-polysaccharides using human fecal inoculum indicates prebiotic potential. Mol. Nutr. Food Res. 62, e1800232 (2018).

    Article  PubMed  Google Scholar 

  43. Fehlbaum, S. et al. In vitro fermentation of selected prebiotics and their effects on the composition and activity of the adult gut microbiota. Int. J. Mol. Sci. 19, 3097 (2018).

    Article  PubMed Central  Google Scholar 

  44. Pérez-Burillo, S. et al. Potential probiotic salami with dietary fiber modulates metabolism and gut microbiota in a human intervention study. J. Funct. Foods 66, 103790 (2020).

    Article  Google Scholar 

  45. Kolodziejczyk, A. A., Zheng, D. & Elinav, E. Diet–microbiota interactions and personalized nutrition. Nat. Rev. Microbiol. 17, 742–753 (2019).

    Article  CAS  PubMed  Google Scholar 

  46. Magnúsdóttir, S. et al. Generation of genome-scale metabolic reconstructions for 773 members of the human gut microbiota. Nat. Biotechnol. 35, 81–89 (2017).

    Article  PubMed  Google Scholar 

  47. Coyte, K. Z. & Rakoff-Nahoum, S. Understanding competition and cooperation within the mammalian gut microbiome. Curr. Biol. 29, R538–R544 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Payne, A. N., Zihler, A., Chassard, C. & Lacroix, C. Advances and perspectives in in vitro human gut fermentation modeling. Trends Biotechnol. 30, 17–25 (2012).

    Article  CAS  PubMed  Google Scholar 

  49. Cohen, S. M. Human relevance of animal carcinogenicity studies. Regul. Toxicol. Pharmacol. 21, 75–80 (1995). discussion 81-86.

    Article  CAS  PubMed  Google Scholar 

  50. Eckburg, P. B. et al. Diversity of the human intestinal microbial flora. Science 308, 1635–1638 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Ley, R. E. et al. Obesity alters gut microbial ecology. Proc. Natl Acad. Sci. USA 102, 11070–11075 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Belkaid, Y. & Hand, T. W. Role of the microbiota in immunity and inflammation. Cell 157, 121–141 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Shankar, V. et al. The networks of human gut microbe-metabolite associations are different between health and irritable bowel syndrome. ISME J. 9, 1899–1903 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  54. Aguirre, M. & Venema, K. Challenges in simulating the human gut for understanding the role of the microbiota in obesity. Benef. Microbes 8, 31–53 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Gratton, J. et al. Optimized sample handling strategy for metabolic profiling of human feces. Anal. Chem. 88, 4661–4668 (2016).

    Article  CAS  PubMed  Google Scholar 

  56. Gorzelak, M. A. et al. Methods for improving human gut microbiome data by reducing variability through sample processing and storage of stool. PLoS ONE 10, e0134802 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Long, W. et al. Differential responses of gut microbiota to the same prebiotic formula in oligotrophic and eutrophic batch fermentation systems. Sci. Rep. 5, 1–11 (2015).

    Article  Google Scholar 

  58. Paliy, O. & Shankar, V. Application of multivariate statistical techniques in microbial ecology. Mol. Ecol. 25, 1032–1057 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Pérez-Burillo, S., Rufián-Henares, J. A. & Pastoriza, S. Effect of home cooking on the antioxidant capacity of vegetables: relationship with Maillard reaction indicators. Food Res. Int. 121, 514–523 (2019).

    Article  PubMed  Google Scholar 

  60. Quince, C., Walker, A. W., Simpson, J. T., Loman, N. J. & Segata, N. Shotgun metagenomics, from sampling to analysis. Nat. Biotechnol. 35, 833–844 (2017).

    Article  CAS  PubMed  Google Scholar 

  61. Morales, F. J., Somoza, V. & Fogliano, V. Physiological relevance of dietary melanoidins. Amino Acids 42, 1097–1109 (2012).

    Article  CAS  PubMed  Google Scholar 

  62. Bauer, E. & Thiele, I. From network analysis to functional metabolic modeling of the human gut microbiota. mSystems 3, 00209–00217 (2018).

    Article  Google Scholar 

  63. Fernandes, J., Su, W., Rahat-Rozenbloom, S., Wolever, T. M. S. & Comelli, E. M. Adiposity, gut microbiota and faecal short chain fatty acids are linked in adult humans. Nutr. Diabetes 4, e121–e121 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Bjerrum, J. T. et al. Metabonomics of human fecal extracts characterize ulcerative colitis, Crohn’s disease and healthy individuals. Metabolomics 11, 122–133 (2015).

    Article  CAS  PubMed  Google Scholar 

  65. Nistal, E. et al. Differences in faecal bacteria populations and faecal bacteria metabolism in healthy adults and celiac disease patients. Biochimie 94, 1724–1729 (2012).

    Article  CAS  PubMed  Google Scholar 

  66. Ze, X., Duncan, S. H., Louis, P. & Flint, H. J. Ruminococcus bromii is a keystone species for the degradation of resistant starch in the human colon. ISME J 6, 1535–1543 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Zhou, L., Xie, M., Yang, F. & Liu, J. Antioxidant activity of high purity blueberry anthocyanins and the effects on human intestinal microbiota. LWT 117, 108621 (2020).

    Article  CAS  Google Scholar 

  68. Gonçalves, G. A. et al. Effects of in vitro gastrointestinal digestion and colonic fermentation on a rosemary (Rosmarinus officinalis L) extract rich in rosmarinic acid. Food Chem. 271, 393–400 (2019).

    Article  PubMed  Google Scholar 

  69. Chen, Y., Chang, S. K. C., Zhang, Y., Hsu, C.-Y. & Nannapaneni, R. Gut microbiota and short chain fatty acid composition as affected by legume type and processing methods as assessed by simulated in vitro digestion assays. Food Chem. 312, 126040 (2020).

    Article  PubMed  Google Scholar 

  70. del Hierro, J. N. et al. In vitro colonic fermentation of saponin-rich extracts from quinoa, lentil, and fenugreek. effect on sapogenins yield and human gut microbiota. J. Agric. Food Chem. 68, 106–116 (2020).

    Article  PubMed  Google Scholar 

  71. Zhang, X. et al. Phytochemical profile, bioactivity, and prebiotic potential of bound phenolics released from rice bran dietary fiber during in vitro gastrointestinal digestion and colonic fermentation. J. Agric. Food Chem. 67, 12796–12805 (2019).

    Article  CAS  PubMed  Google Scholar 

  72. Wang, M. et al. Purified fraction of polysaccharides from Fuzhuan brick tea modulates the composition and metabolism of gut microbiota in anaerobic fermentation in vitro. Int. J. Biol. Macromol. 140, 858–870 (2019).

    Article  CAS  PubMed  Google Scholar 

  73. Rui, Y. et al. Simulated digestion and fermentation in vitro by human gut microbiota of intra- and extra-cellular polysaccharides from Aspergillus cristatus. LWT 116, 108508 (2019).

    Article  CAS  Google Scholar 

  74. Chen, L. et al. Simulated digestion and fermentation in vitro by human gut microbiota of polysaccharides from Helicteres angustifolia L. Int. J. Biol. Macromol. 141, 1065–1071 (2019).

    Article  CAS  PubMed  Google Scholar 

  75. Huang, F. et al. Structural characterization and in vitro gastrointestinal digestion and fermentation of litchi polysaccharide. Int. J. Biol. Macromol. 140, 965–972 (2019).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported by the EU project Stance4Health (contract no. 816303) and by the Plan propio de Investigación y Transferencia of the University of Granada under the program ‘Intensificación de la Investigación, modalidad B’ to J.A.R.-H.

Author information

Authors and Affiliations

Authors

Contributions

S.P.-B., S.M., and J.A.R.-H. wrote the manuscript. B.N.-P., A.J.V.-M., D.H.-N., A.L.-M. and S.P. contributed to the writing of the manuscript. S.P.-B., S.M., B.N.-P., A.J.V.-M., D.H.-N. and A.L.-M. contributed to formal analysis and investigation; S.P.-B. developed the methodology. S.P. and J.A.R.-H. supervised the work. J.A.R.-H. obtained funding and coordinates the EU project Stance4Health.

Corresponding author

Correspondence to José Ángel Rufián-Henares.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Francisco Tomás-Barberán and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Pérez-Burillo, S. et al. Food Chem. 239, 1253–1262 (2018): https://doi.org/10.1016/j.foodchem.2017.07.024

Pérez-Burillo, S. et al. Food Chem. 279, 252–259 (2019): https://doi.org/10.1016/j.foodchem.2018.11.137

Pérez-Burillo, S., et al. Food Chem. 316, 126309 (2020): https://doi.org/10.1016/j.foodchem.2020.126309

This protocol is an extension to: Nat. Protoc. 14, 991–1014 (2019): https://doi.org/10.1038/s41596-018-0119-1

Supplementary information

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pérez-Burillo, S., Molino, S., Navajas-Porras, B. et al. An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality. Nat Protoc 16, 3186–3209 (2021). https://doi.org/10.1038/s41596-021-00537-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-021-00537-x

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing