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

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 extension of a previous in vitro digestion protocol provides a subsequent in vitro batch fermentation stage that is carried out afterward to enable investigation of the effect of food on the gut microbiome.

Automated summary

This protocol describes an in vitro batch fermentation procedure to study the effect of food on gut microbiota. The procedure involves the use of an oligotrophic fermentation medium and a high concentration of fecal inoculum from human fecal samples.