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Objectives To test the effect of probiotic strains (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12), a prebiotic (bacteriophage blend), and a postbiotic (tributyrin) on the human intestinal microbiota in vitro. Methods Short term colonic incubation was performed under conditions representative of the proximal large intestine, using the fecal microbiota of a single healthy human donor. At the start of the experiment, treatments and a blank control were added to the reactors. Each reactor also received a carbohydrate-supplemented nutritional medium and freshly prepared fecal inoculum. Reactors were closed with a rubber septum, made anaerobic through flushing with nitrogen, and incubated for 48 hours. Each condition was run in triplicate. Changes in microbial community composition were determined via 16S-targeted Illumina sequencing and flow cytometry. Enriched bacterial genera were identified using LEfSe (LDA > 2, p < 0.05). Results A significant increase in alpha-diversity was observed by 48h compared to the control. LEfSe revealed that community shifts were attributed to a wide range of bacterial genera including Gemmiger, Lactobacillus, Bacteroides, Eubacterium, Oscillibacter, and Holdemanella at 24h and of genera such as Lactobacillus, Bacteroides, Muribaculum, and Holdemanella at 48h. LGG and BB-12 were not detected in the donor microbiota originally but abundance of both was higher after incubation. Conclusions The results from this study suggest that a combination of pre-, pro-, and postbiotics modulates gut microbiota by increasing microbial diversity and the relative abundance of a number of bacterial genera. An increase in the abundance of LGG and BB-12 indicates growth of these strains in the presence of a human gut microbiota. Future human clinical studies are needed to further investigate the observed effects. Funding Sources The study was conducted by an independent lab, ProDigest, and funded and sponsored by Ritual (Natals, Inc.). Compound Solutions, Inc. also financially supported the study.

Objectives To test the effects of a novel product combining probiotics (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12), a prebiotic (bacteriophage blend), and a postbiotic (tributyrin), and its individual components, on intestinal permeability in an in vitro gut barrier model. Methods Short term colonic incubation was performed for the individual components, the full product, and a blank control under conditions representative of the proximal large intestine, using the fecal microbiota of a single healthy human donor. Colonic batch suspensions were collected after 48 hours of incubation and filter sterilized. Caco-2 cell monolayers were placed on top of PMA-differentiated THP1-BlueTM cells, creating an in vitro model of intestinal barrier disruption. Colonic fermentation samples were diluted in complete medium and given apically to Caco-2/THP1-BlueTM co-cultures for 24 hours. Intestinal permeability was assessed with transepithelial electrical resistance (TEER). TEER was measured in Caco-2 monolayers at baseline and 24 hours after pre-treatment of Caco-2/THP-1 BlueTM co-cultures. Each final TEER value was normalized to its corresponding initial value. Results Application of colonic suspensions from the combination of pre-, pro-, and postbiotics significantly increased TEER values compared to control (p < 0.05). Probiotics and tributyrin were also found to increase TEER (p < 0.05) but no significant effect was observed for prebiotic phages (p > 0.05). Conclusions Colonic batch fermentations of a novel combination of pre-, pro-, and postbiotics showed improved gut barrier function in vitro, indicated by increased TEER values. Results suggest that probiotic strains and tributyrin likely modulate these effects, consistent with previous research linking the probiotic LGG and short chain fatty acid butyrate to gut barrier protection. Funding Sources The study was conducted by an independent lab, ProDigest, and funded and sponsored by Ritual (Natals, Inc.). Compound Solutions, Inc. also financially supported the study.

Objectives To test the survival and release of probiotics (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12), a prebiotic (bacteriophage blend), and a postbiotic (tributyrin) in a capsule-within-capsule design during passage through the gastrointestinal tract under fed and fasted conditions. Methods Using an adapted Simulator of the Human Intestinal Microbial Ecosystem (SHIME®) model, physiological conditions of the stomach and small intestine, including pH profiles, temperature, incubation time, and addition of bile liquid and digestive enzymes, were simulated within the same reactor over time. The capsule contents included an inner capsule, containing probiotics and prebiotic phages, and an outer capsule, containing the postbiotic, with different delivery and release characteristics. Probiotic survival was assessed via plate counts and PMA-qPCR, release of phages was assessed via phage titer assay, and tributyrin release was assessed via gas chromatography. All experiments were performed in triplicate to account for biological variability. Results Under fasted conditions, the majority of the probiotics bypassed the stomach and small intestine in a viable form, of which more than 11 billion CFU were culturable. There were no significant differences between fasted and fed states. Similarly, the majority of the prebiotic phages survived transit through the stomach and small intestine. Tributyrin was mostly released in the small intestine. Conclusions The capsule design ensured probiotics and prebiotic phages survived the harsh conditions of the upper gastrointestinal tract for delivery to the colon. The release of tributyrin within the small intestine supports its breakdown by pancreatic enzymes to produce short chain fatty acid butyrate. The findings of this study suggest a novel, colon-targeted probiotic delivery system explained by specific capsule and content characteristics. Funding Sources The study was conducted by an independent lab, ProDigest, and funded and sponsored by Ritual (Natals, Inc.). Compound Solutions, Inc. also financially supported the study.