In Vitro Examination of the Effect of Honey in the Gastrointestinal Tract

Abstract

Objectives Honey has shown beneficial antibacterial properties in wound healing, but little is known about the effects of honey consumption on human gut health. Herein, we investigate the effects of honey gastric digest on the growth of the common food-borne pathogen Enterotoxigenic E. coli (ETEC) and beneficial lactic acid bacteria within the gastric environment and a small intestine microbial community, respectively. Methods In Vitro Gastric Digestion: An in vitro digestion of both honey and a simple sugar control (42% fructose, 35% glucose, 23% water) was performed in a simulated gastric electrolyte solution containing pepsin and hydrochloric acid (HCl). Each solution was adjusted to a pH of 2.5, and placed in an incubating shaker at 37°C, 200rpm for 120 minutes. For ETEC survival experiments, 800,000 CFU/mL of ETEC in brain heart infusion (BHI) broth was added to each solution before pH adjustment. Samples were collected every 20 minutes, plated on BHI, and colonies counted the following morning. In Vitro Fermentation: Digested honey or control was added at 1% w/v to a small intestine microbial mock community, containing 100,000 cells each of 8 common small intestinal commensal bacteria, and fermented anaerobically for 40 hours at 37°C and an initial pH of 5.5. Samples were collected for 16S rRNA analysis; optical density and pH were measured to determine bacterial growth and estimate the relative production of acids in the culture media, respectively. 16S Analysis: For individual CFU, full length amplicons were analyzed by Sanger sequencing and NCBI BLAST. For fermentations, the V4 region was amplified and sequenced on Illumina MiSeq at UC Davis Tech Core. Sequences were analyzed in Qiime 2. Statistical analyses were performed via DESeq2 in R. Results Our preliminary data shows that under in vitro gastric digestion conditions, ETEC survival decreased more quickly in honey than a simple sugar control. Total CFU were similar between the two conditions at 120 minutes, but Sanger sequencing identified a majority of colonies plated from the honey condition as genus Bacillus. During in vitro fermentation, differences in the relative abundance of specific taxa between honey and control did not reach statistical significance. Conclusions Our pilot experiments suggest honey may affect the survival of gastric pathogens within the stomach. Research is ongoing to confirm these results. Funding Sources The National Honey Board, USDA, and the Dean's Distinguished Graduate Fellowship, UC Davis.