Introduction The prevalence of plastic particles in the food chain is already evident. Therefore, the scientific community is concerned about the health risks of food-use microplastics; although the risk assessment of microplastics is not possible, it is a current global challenge. The dynamic gastrointestinal simulator (simgi®) pursues the need for a dynamic in vitro simulation of the human gastrointestinal tract adapted to food safety and health fields (www.cial.uam-csic.es/simgi/). The simgi® system can simulate separately or in continuous gastric, intestinal digestion, and colonic fermentation, under computer control of the physicochemical parameters. Furthermore, the simgi® platform has confirmed its models suitability since its first studies showed the consistency between the simulated digestion of food products and the metabolic evolution observed with the intake of the same products in human intervention studies. Its modular configuration has allowed the study of miscellaneous products as dietary fibres, probiotics, proteins and polyphenol-rich food matrices to evaluate their gastrointestinal digestion and impact on gut microbiota. Moreover, simgi® dynamic operation allows the simulation of acute and chronic intakes, which are used to study short- and medium-term effects of digested products. Therefore, the simgi® system is an exclusive tool to monitor avant-garde products of interest, such as nanoparticles [1] and microplastics, which food safety is yet to determine. This study aims to evaluate the impact of the main stages of the human gastrointestinal tract on different relevant food-use plastic materials, also to ascertain the effect of digested microplastics on gut microbiota composition and functionality. Methodology Two microplastic size ranges have been used as prototypes: pellets between 2.8 and 3.6 mm and milled plastics between 50 and 300 µm. Polyethylene terephthalate (PET) was considered as a model for non-biodegradable microplastic, whereas polylactic acid (PLA) was used as biodegradable. Initial steps focused on performing the studies needed to configure the simgi® system as a useful tool for monitoring microplastics in the human gastrointestinal tract. Thus PET pellets, milled PET, PLA pellets, and milled PLA were digested in vitro according to the standardised static gastrointestinal digestion food protocol [2] to establish digestion influence on the microplastics and simgi® digestion parameters. Microplastics size, shape, and stability (using FESEM, FT-IR Spectroscopy, and Raman spectra) were evaluated after gastric and small intestinal digestion for the microplastic models. The physiological conditions of the ascending, transverse, and descending colon were reproduced sequentially on the simgi® colonic bioreactors, which included a simulated human colonic microbial community, to study the interaction between the gut microbiota and microplastics. Each colonic bioreactor was fed with a dose of 0.166 mg digested milled PET particles. The milled PET dose, was selected considering previous estimations of human microplastic intake from food and beverages [3]. Changes in microbiota composition (microbial plate counting and sequencing 16S rRNA), microbial metabolic activity (ammonium and SCFA production), and microplastics shape and stability (using FESEM, FT-IR Spectroscopy, and Raman spectra) were evaluated in the ascending, transverse, and descending colon after feeding the system with microplastics. Results In this study, a novel lab practical guidance, designed for operation with microplastics, on simgi® was proposed. At the gastric and intestinal levels, there were no remarkable changes for the size or structure of the studied particles. However, all microplastics showed organic material deposits on their surface after in vitro gastrointestinal digestions (Figure 1 as an example of milled PET after gastric and intestinal digestion). At the colonic level, milled PET particles induced several changes in colonic microbiota composition/diversity. Using plate counting, total aerobic and total anaerobic microorganisms considerably decreased (Δ log (CFU/mL) ≥- 3) during 72 hours of colonic fermentation in all colonic compartments. Furthermore, a notable reduction of Bifidobacterium spp. and Clostridium spp. groups were observed after the first 48 hours of fermentation. Likewise, 16S rRNA gene sequence analysis showed reductions in alpha-diversity indexes, especially for the transverse and descending colon in the first 24 hours of fermentation. Phylogenetic analysis at the phylum level exhibited differences in the relative abundances of important taxa after being fed microplastic, although they were compartment dependent. Bacteroidetes was reduced for all colonic compartments, whereas Firmicutes abundance increased in the ascending colon, Proteobacteria and Synergistetes in the transverse colon, and Desulfobacteria in the descending colon. Regarding the most important members at the genus level, results revealed differences in the relative levels of Bacteroides and Parabacteroides, with an important decrease in all colonic compartments being detected, and the most evident effect in transverse and descending colon, reaching values below 5%. Discussion In vitro static gastrointestinal digestion simulations [2] allowed the study of the interaction between microplastics and the human gastrointestinal tract. Furthermore, results presented at the gastrointestinal level, agree with those reported for microplastic gastrointestinal digestions [4], suggesting the formation of an organic corona in particles by the simulated digestive fluids. However, neither the colonic stage nor colonic microbiota are included in previous digestion models, which also cannot simulate the effects of a chronic intake, which is the actual situation of microplastics intake, considering the latest data estimated for the inadvertent ingestion of microplastics [3]. Therefore, it is noteworthy to be able to simulate the colonic stages, as it was simulated on the dynamic simgi® system. In fact, this study has evaluated for the first time, to the best of our knowledge, the microplastic effect on the human colonic microbiota. Colonic bacterial populations showed a decrease in alpha-diversity and Firmicutes/Bacteroidetes ratio, which are considered as health-related indexes. Furthermore, the analysis of the key members of the microbiota at family and genus levels corroborated the results which point to a possible intestinal dysbiosis after being fed microplastics, as was reported for mice models [5]. Although this project is still in its infancy, the simgi® platform appears a useful tool to study the effects of microplastics along the gastrointestinal tract, and an effective support on research and food industry development by acting complementary and/or as a previous level to human studies, given their ethical and economic restrictions. References [1] Cueva, C., Gil-Sánchez, I., Tamargo, A., Miralles, B., Crespo, J., Bartolomé, B., & Moreno-Arribas, M. V. V. (2019). Gastrointestinal digestion of food-use silver nanoparticles in the dynamic SIMulator of the GastroIntestinal tract (simgi®). Impact on human gut microbiota. Food and Chemical Toxicology, 132(July), 110657. https://doi.org/10.1016/j.fct.2019.110657 [2] Brodkorb, A., Egger, L., Alminger, M., Alvito, P., Assunção, R., Ballance, S., Bohn, T., Bourlieu-Lacanal, C., Boutrou, R., Carrière, F., Clemente, A., Corredig, M., Dupont, D., Dufour, C., Edwards, C., Golding, M., Karakaya, S., Kirkhus, B., Le Feunteun, S., … Recio, I. (2019). INFOGEST static in vitro simulation of gastrointestinal food digestion. Nature Protocols, 14(4), 991–1014. https://doi.org/10.1038/s41596-018-0119-1 [3] Senathirajah, K., Attwood, S., Bhagwat, G., Carbery, M., Wilson, S., & Palanisami, T. (2021). Estimation of the mass of microplastics ingested – A pivotal first step towards human health risk assessment. Journal of Hazardous Materials, 404(PB), 124004. https://doi.org/10.1016/j.jhazmat.2020.124004 [4] Stock, V., Fahrenson, C., Thuenemann, A., Dönmez, M. H., Voss, L., Böhmert, L., Braeuning, A., Lampen, A., & Sieg, H. (2020). Impact of artificial digestion on the sizes and shapes of microplastic particles. Food and Chemical Toxicology, 135(October 2019). https://doi.org/10.1016/j.fct.2019.111010 [5] Luo, T., Wang, C., Pan, Z., Jin, C., Fu, Z., & Jin, Y. (2019). Maternal Polystyrene Microplastic Exposure during Gestation and Lactation Altered Metabolic Homeostasis in the Dams and Their F1 and F2 Offspring. Environmental Science and Technology, 53(18), 10978–10992. https://doi.org/10.1021/acs.est.9b03191 Figures Figure 1. FESEM images to evaluate the milled PET surface after in vitro gastrointestinal digestion.