Culture-dependent and independent approaches for evaluating the impact of stilbenoids on the gut microbiota

Stilbenoids, a group of plant phenolic compounds, may have multiple biological benefits, such as anti-inflammation, anticancer and antimicrobial activity. Also, they are dietary phenolics that occur in a wide range of edible fruits and seeds. It follows that bacteria of the gut microbiota are naturally exposed to different amount of these compounds. The aim of this study was the investigation of the possible effect of pterostilbene and viniferinfuran, resveratrol derivates, in affecting the overall viability of fecal samples and the culturability of specific groups of bacteria (i.e., Bifidobacteria and mucin-degrading bacteria) belonging to the gut microbiota. Fecal samples from 5 healthy donors were collected, and exposed to selected polyphenols for 3h at 37°C. Then, the overall viability was assessed by flow cytometry by staining fecal cell suspensions with SYTO 24 and propidium iodide in order to distinguish Active Fluorescent Unites (AFUs, considered as live cells) and non-Active Fluorescent Units (non-AFUs, considered as dead cells). Also, selective media were used for investigating some relevant classes of microorganisms for GUT microbiota such as the genus of Bifidobacterium spp and mucin-degrading bacteria (i.e., Akkermansia muciniphila). By flow cytometry we measured a decreasing AFU in AFU population by increasing Pterostilbene or Viniferinfuran (28μg/ml) concentrations in all the tested samples. Pre-treatments of the fecal samples with NaOH-SDS for the removal of Gram-negative bacteria confirmed that the most sensitive bacteria towards stilbenoids belong to the Gram-positive group. These results were confirmed also by a culture-dependent method by using selective media for Bifidobacteria and mucin-degrading bacteria.