Clover honey limits survival of enterotoxigenic Escherichia coli in an in vitro gastric model and reduces in vitro fermentation pH of small intestine microbes

Authors: TRAXLER, STE - University Of California, Davis; Storms, David; Faso, Alyce; Alkan, Zeynep; BOWLUS, CHRISTOPHER - University Of California, Davis; Lemay, Danielle; Kable, Mary

Submitted to: Journal of Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/6/2026
Publication Date: 4/8/2026
Citation: Traxler, S., Storms, D.H., Faso, A.R., Alkan, Z., Bowlus, C., Lemay, D.G., Kable, M.E. 2026. Clover honey limits survival of enterotoxigenic Escherichia coli in an in vitro gastric model and reduces in vitro fermentation pH of small intestine microbes. Journal of Nutrition. 156(6). https://doi.org/10.1016/j.tjnut.2026.101530.
DOI: https://doi.org/10.1016/j.tjnut.2026.101530

Interpretive Summary: Very few studies have assessed how honey might interact with our stomach and gut microbes during consumption. We used a laboratory model of the human stomach and small intestine to examine the digestion of clover honey relative to a matched sugar control. We found that at a pH of 2.5, which is typical for a healthy human stomach in a fasted state, honey can inhibit the growth of food-borne pathogen, Enterotoxigenic E. coli, to a greater extent than an equivalent amount of sugar (fructose and glucose). Fermentation of digested honey by microbes collected from distal human small intestines during routine colonoscopy, caused a greater decrease in pH than metabolism of sugar by the same microbes. Additionally when the microbial fermentation products were applied to cultured cells from the lining of the colon (Caco-2 cells), honey fermentation products tended to improve barrier function to a greater extent than sugar control fermentation products.

Technical Abstract: Objectives: Honey has well described antimicrobial properties in wound healing, but little is known about the effects of honey within the gastrointestinal tract. We used in vitro gastric digestion and fermentation of clover honey, with small intestine bacteria (SIB), to investigate its impacts on microbial community composition, intestinal epithelial cell health and inhibition of the foodborne pathogen ETEC H10407. Methods: In vitro gastric digestions of sterile filtered honey, and a simple sugar control (42% fructose, 35% glucose, 23% water) were performed in a simulated gastric electrolyte solution containing pepsin and hydrochloric acid (HCl) spiked with 1x108 CFU/mL ETEC strain H10407. The pH and ETEC CFUs were measured. Resulting digesta were added at 1% w/v to batch fermentations with SIB, in the form of a mock community or ileal aspirate from 6 healthy donors and fermented anaerobically for 6 hours at 37' C and an initial pH of 5.8. Microbial composition changes were determined via 16S rRNA amplicon sequencing. Fermentation supernatants were applied to mature Caco2 cell monolayers in Transwell plates for analysis of changes in transepithelial electrical resistance and IL-8 production. Results: Under in vitro gastric digestion conditions ETEC H10407 survival decreased significantly more in the presence of clover honey than a simple sugar control. Low pH was necessary for this effect. No significant effects on microbial community composition with or without the presence of a pathogen were observed. However, the pH of fermentation media was significantly reduced with honey relative to sugar control. Honey fermentation supernatant from human ileal communities challenged with ETEC elicited a greater improvement in TEER relative to the same fermentations performed with a control. Conclusion: Honey limits the survival of ETEC H10407 within an in vitro gastric environment at low pH. Clover honey may also impart more significant decreases in pH and potential improvement in gastrointestinal barrier function in the presence of a pathogen.