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Abstract

Abstract In vitro models simulating the human gut are useful tools for understanding the complexity of the gastrointestinal microbiome and its interactions with food components and other environmental factors. However, existing colon models are either relatively simple, leaving important parameters uncontrolled (such as colonic pH), or suffer from low throughput. Here, we introduce the HUGS (High-throughput Universal Gut Simulator) protocol for simulating colonic passage in vitro , offering a high-throughput approach for studying gut microbiome dynamics. By replicating ascending colon conditions, including controlled pH, anaerobicity, and temperature, we aimed to simulate the gut environment while maintaining experimental scalability. The microbiome modulatory effects of fermented rapeseed meal, soybean meal, seaweed, and different xylans, both individually and in various combinations, were evaluated using the HUGS protocol. The resulting in vitro colonic microbiome composition was determined by 16 S rRNA gene amplicon sequencing, while short-chain fatty acid (SCFA) production was quantified by ion-exchange chromatography to assess microbial metabolic activity. During a single 24-hour run, HUGS enables 32 individual fermentations to be performed simultaneously, each with independent pH control under anaerobic conditions. The model supported the growth of key anaerobic gut microorganisms, including Faecalibacterium prausnitzii and Akkermansia muciniphila , while preserving the dominant phylum-level characteristics of the original fecal microbiomes. Although alpha diversity decreased following fermentation, as commonly observed in short-term in vitro colon models, obligate anaerobic taxa remained abundant throughout the experiments. Overall substrate treatments did not produce distinct clustering patterns. However, differential abundance analysis identified substrate-dependent shifts in specific taxa, including increased relative abundance of Akkermansia muciniphila and Leuconostoc citreum , together with reduced abundance of Cutibacterium acnes and Clostridium perfringens in fermentations containing fermented plant materials. SCFA production varied between substrate formulations, including a significant reduction in total SCFA concentration (p-adj. = 0.016) following xylan supplementation of the fermented soybean meal and seaweed formulation. These findings demonstrate that the HUGS in vitro colon model combines physiologically relevant environmental control with substantially increased throughput compared to existing colon simulation platforms. By supporting growth of key anaerobic gut microorganisms while maintaining sensitivity to biologically meaningful substrate-dependent responses, HUGS is proposed a promising platform for high-throughput screening of dietary fibers, feed additives, and microbiome-modulating compounds in both fundamental and translational future research.