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Gut microbes interact with the host in multiple ways. One of the most important functions of microbes is their metabolism capacity. Through gut microbial metabolism, chemicals in the environment or in the food can be transformed into interesting products. The metabolism reactions help human hosts in many ways: In the case of toxic environmental xenobiotics, products that are less toxic are formed; and in the case of food components, more bioaccessible metabolites are produced. For example, most dietary fibers are indigestible in human intestines, and they can be broken down with the help of gut microbiota. Another example of food components that need the help of gut microbiota is polyphenol. Polyphenols are known to be good antioxidants that exhibit many health impacts. However, their health functionalities are limited due to their structure related low bioaccessibility. Gut microbiota modifies polyphenols through a variety of phase I metabolism reactions. The gut microbiota mediated polyphenol metabolites play an important role in the health impacts of the polyphenols. Differential gut microbiota and metabolites help explain the differential individual responses to diets and polyphenol supplements. Many methylated polyphenols were studied for their bioactivities. Representatives are pterostilbene (PTE) from blueberries, curcumin (CUR) from turmeric and PMF (polymethoxyflavones) from citrus fruits like tangeretin (TAN) and nobiletin (NOB). The benefits of consuming these phytochemicals are implicated in many diseases such as IBD (inflammatory bowel disease), CVD (cardiovascular diseases), type II diabetes, and cancers. Gut microbiota alterations were often observed to be associated with the abovementioned diseases and foods. The objective of this study is to investigate the role of gut microbiota in the biofunctionalities of the four methylated phytochemicals by looking at both the gut microbial metabolism mechanisms and the bioactivities of microbial metabolites. Firstly, we screened for the gut microbiota single strain that can metabolize the selected phytochemicals PTE, CUR, TAN, and NOB using a modified culture medium for bacteria. As found by former researchers in our group, O-demethylated metabolites of the phytochemicals are highly likely associated with gut microbiota. Therefore, commercial gut microbiota strains that were reported to have demethylation capacity against other substrates are obtained and screened for their metabolizing capacity on our selected phytochemicals. Among all the screened single strains, the strain Eubacterium limosum DSM 20543 can metabolize all four phytochemicals through O-demethylation and reduction. Secondly, we characterized the demethylase in Eubacterium limosum DSM 20543 which is responsible for the demethylation of PTE and PMF using genetic methods. By comparing the transcriptome of E. limosum treated with or without phytochemicals, putative demethylase genes were selected and cloned for in vitro function validation. With the formation of metabolites confirmed with LC-MS, the enzyme genes were determined. The protein structure of the four-component O-demethylase was predicted with bioinformatics tools. Thirdly, we investigated the role of Eubacterium limosum DSM 20543 in the in vivo metabolism of phytochemicals using a mouse model. We used antibiotics to eliminate the gut microbiome in mice before giving E. limosum to them. Phytochemicals TAN or CUR were given to mice through oral gavage, and waste samples were collected at different time points for metabolomic studies. At the end of the study, the GI (gastrointestinal) tracts of mice were collected for metabolomic studies. After comparison, the group of mice that received E. limosum had more demethylated metabolites of the corresponding phytochemical, indicating the importance of E. limosum in the metabolism of TAN and CUR. Lastly, the bioactivities of E. limosum metabolites were determined using cell models. Metabolite cocktails of E. limosum were collected and extracted with ethyl acetate after in vitro fermentation of the bacteria and the four target phytochemicals. Anti-inflammatory and anti-cancer properties were determined with RAW 264.7 and HCT 116 cells. Among all the time points and phytochemicals, the metabolite mixture of TAN at 72-hour fermentation exhibited the strongest bioactivities. Overall, we used a combination of metabolomic and genomic methods to investigate the role of gut microbiota in the metabolism and health impacts of methylated phytochemicals. The strain E. limosum DSM 20543 can metabolize the phytochemicals with a novel O-demethylase to produce metabolites with bioactivities and alter the metabolomic profiles of the phytochemicals in mice. These results emphasize the importance of gut microorganisms in the health functionalities of diet.
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