An in vitro batch fermentation protocol for studying the contribution of food to gut microbiota composition and functionality Pérez Burillo, Sergio Molino, Silvia Navajas Porras, Beatriz Valverde Moya, Álvaro Jesús Hinojosa Nogueira, Daniel José López Maldonado, Alicia Pastoriza de la Cueva, Silvia Rufián Henares, José Ángel Microbiome Nutrition Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41596-021-00537-x. 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. 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. 2022-01-12T09:55:59Z 2022-01-12T09:55:59Z 2021 journal 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] http://hdl.handle.net/10481/72320 10.1038/s41596-021-00537-x eng http://creativecommons.org/licenses/by-nc-nd/3.0/ open access Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License