The gastrointestinal tract (GIT) is an organ system responsible for food digestion,absorption of nutrients and the expelling of waste. Due to a high demand for testing theintestinal faith of pharmaceutical and bioactive food formulations, there is great interestfrom food, bioscience and pharmaceutical industries to simulate this complex system.Human intervention studies are still the golden standard for digestion simulation, but theyare hampered by high costs, difficulties in accessing samples (especially for continuousmonitoring) and ethical constrains. To address these issues, different in vivo models ofanimals have been developed. Despite the many advantages, they have serious drawbacks,since results obtained by such methods cannot be directly extrapolated to humans.Moreover, according to the 3Rs framework (Replacement, Reduction and Refinement), theusage of animals in experimental studies should be reduced and, if possible, replaced.Consequently, a variety of in vitro GIT models have been developed, which aim atcircumventing these challenges. However, all existing dynamic in vitro GIT models arelimited by high working volumes and low throughput, which renders any screening effortvirtually impossible, and the lack of technical replicates reduces the statistical strength ofresults. This work presents current knowledge about available GIT simulation tools and areview on existing in vitro models. Furthermore, it describes the development of a lowvolume increased throughput in vitro model of the small intestine (duodenum, jejunum,ileum) as a screening platform for the study of food digestibility, intestinal survival ofprobiotics and absorption of drugs and small nutrients.The GIT harbours a vast number of microorganisms called the gut microbiota.These bacteria are unevenly distributed along the GIT, ranging from 101-103 cells/g in thestomach, through 103-108 cells/g in the small intestine and up to 1012 cells/g in the colon.In the last decade, numerous studies have been conducted focussing on the faecalmicrobiota composition and its impact on the host health. The microbiota inhabiting thesmall intestine was largely overlooked, although recent studies imply it might play a majorrole in digestion processes. One of the focal areas of this project was hence to develop asimulated small intestine microbiota consortium to be incorporated into the in vitro modelof the small intestine.In order to evaluate the usefulness of the developed in vitro model for simulatingthe small intestine passage, the survival of three Lactobacillus sp. strains was tested, andthe obtained results were compared with previously reported values. We also studied howdifferent feeding conditions (fed, fasted) and the presence of small intestine microbiotainfluence intestine persistence of probiotic bacteria. In the same manuscript, we describedfor the first time the fully functional in vitro model prototype called “The SmallestIntestine (TSI)”. The model proved to be a cost-efficient, fast and reproducible method forthe simulation of the passage through the small intestine.Subsequently, TSI was implemented to investigate the viability ofmicroencapsulated Akkermansia muciniphila and Lactobacillus plantarum during passagethrough in vitro simulated upper GIT. The survival of coated and naked cells in the smallintestine was investigated in both fed and fasted conditions in the TSI model. Resultsindicated a protective effect of xanthan/gellan gum for L. plantarum and decreasedviability of coated A. muciniphila due to the desiccation effect of coating, which probablycaused leakage of the cell membrane.In another study, we investigated the persistence and performance of abacteriophage cocktail targeting Escherichia coli DSM 1058 in the small intestine andcompared its impact on the simulated small intestine microbiota with a broad-spectrumantibiotic. Tested bacteriophages were not affected by bile salts or pancreatic juice andmaintained their efficiency towards the targeted bacteria. Moreover, the phage cocktailhad only minor impact on the small intestine microbiota, contrary to the antibiotic, whichcaused collateral damage killing 90% of all bacteria.In the fourth manuscript, we presented the flexibility of the TSI model by adoptingit to simulate the passage through the upper intestine (stomach and small intestine) of apiglet. Following these adjustments, we tested intestinal behaviour and survival of threeBacillus sp. strains in the form of spores and vegetative cells. We reported sufficientsurvival of bacilli spores and, for some strains, observed a round of germination and resporulationduring the passage. Moreover, we proved that Bacillus sp. need to beadministrated in the form of spores to reach their site of action in the small intestine.A comprehensive understanding of the challenges associated with the modelling ofthe small intestine conditions as well as advantages and weaknesses of implementation ofin vitro models are presented in this dissertation. The development of the TSI model is thenext step towards a simple and cost-efficient benchtop GIT model with high screeningpossibilities, which will allow reducing the usage of live animals in experimental studies.