# In Vitro Simulation of Human Colonic Fermentation:
## 5 Addressing Colonic In Vitro Digestion Studies
### Changes in the Microbiota Populations and Their Metabolic Response  
Either during the fermentation process or at the endpoint, samples are collected from the reactors to monitor the effect of the experimental conditions on the microbial populations and activity, which also referred to as the basal situation after inoculum stabilisation. Among them, the composition of microbial populations and the production of metabolites are of great importance when assessing the impact of food or extracts on the gut microbiota. In this context, metaomic approaches, which include metagenomics, metatranscriptomics, metaproteomics, and metabolomics have been developed and widely applied to characterize microbial communities [107]. Additionally, some other techniques are applied depending on study aim and tools availability. Metagenomic techniques, both 16S rRNA sequencing and shotgun metagenomics, have been widely applied to determine the composition and functionality of human microbial communities. Nevertheless, metagenomics cannot elucidate which genes are expressed. In this context, metotranscriptomic and metaproteomic methods have gained weight. On the one hand, metotranscriptomic methods, which study the transcripts produced by a microbial community, reveal knowledge related to the induced/repressed genes and active/inert/dead microorganisms. The tools used for metagenomics assessment can be adapted to be used in metotranscriptomics methods, which allow for its easy integration. On the other hand, metaproteomic methods contribute to a better understanding of microbial functionality since they study the proteins produced by a microbial community. In this case, mass spectrometry (MS) is generally performed. Finally, in terms of functionality assessment, the metabolomic approach studies the metabolic pattern of a microbial community. Both mass spectrometry (MS) and nuclear magnetic resonance (NMR) are applied, but MS is preferred over NMR because of its higher sensibility. Even though it is still challenging, the integration of metaomic data could bring promising insights in terms of the human microbiota composition, functionality, and metabolite activity [108]. After this introduction, some of the analytical techniques used to assess in vitro gut fermentation in the last ten years were collected. Regarding the analysis of microbiota populations, 16S rRNA gene sequencing is widely applied [36; 37; 54]. In parallel, other methodologies can be used to target or quantify specific bacteria genera or species or total bacteria, depending on the specificity of the primer used (e.g., qPCR, PCR-DGGE gel or FISH) [26; 53; 80; 102]. When it comes to metabolite analysis, SCFA or BCFA production are typically analysed using gas or liquid chromatography (GC or LC) coupled with either different detectors (e.g., FID or UV) or MS [43; 54; 56], but when other metabolites, such as phenolic compounds, are being considered in the studies, LC techniques coupled with MS [44; 102] or antioxidant tests are commonly used when studying antioxidant capacity [33]. Finally, ammonium, pH, lactic acid, and gas volume measurements are also analysed in most of the studies [36; 65; 71]. Table 3 collects different analytical techniques that usually applied in in vitro gut fermentation studies.