Development of fecal microbial enzyme mix for mutagenicity assay of natural products
Abstract
Orally administered herbal glycosides are metabolized to their hydrophobic compounds by intestinal microflora in the intestine of animals and human, not liver enzymes, and absorbed from the intestine to the blood. Of these metabolites, some, such as quercetin and kaempherol, are mutagenic. The fecal bacterial enzyme fraction (fecalase) of human or animals has been used for measuring the mutagenicity of dietary glycosides. However, the fecalase activity between individuals is significantly different and its preparation is laborious and odious. Therefore, we developed a fecal microbial enzyme mix (FM) usable in the Ames test to remediate the fluctuated reaction system activating natural glycosides to mutagens. We selected, cultured, and mixed 4 bacteria highly producing glycosidase activities based on a cell-free extract of feces (fecalase) from 100 healthy Korean volunteers. When the mutagenicities of rutin and methanol extract of the flos of Sophora japonica L. (SFME), of which the major constituent is rutin, towards Salmonella typhimurium strains TA 98, 100, 102, 1,535, and 1,537 were tested using FM and/or S9 mix, these agents were potently mutagenic. These mutagenicities using FM were not significantly different compared with those using Korean fecalase. SFME and rutin were potently mutagenic in the test when these were treated with fecalase or FM in the presence of S9 mix, followed by those treated with S9 mix alone and those with fecalase or FM. Freeze-dried FM was more stable in storage than fecalase. Based on these findings, FM could be usable instead of human fecalase in the Ames test.
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Concerns about variability of the fecal incubation assay have led to development of a fecal microbial enzyme mix.
“Concerns about the variability of the assay have seen the development of a fecal microbial enzyme mix (Yeo et al., 2012).”
From Gut reactions: Breaking down xenobiotic–microbiome interactions (2019) · cites this paper as Yeo et al., 2012
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“Concerns about the variability of the assay have seen the development of a fecal microbial enzyme mix (Yeo et al., 2012).”
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Kang, M. J., Kim, H. G., Kim, J. S., Oh, D. G., Um, Y. J., Seo, C. S., Han, J. W., Cho, H. J., Kim, G. H., Jeong, T. C., et al. (2013) · Expert Opinion on Drug Metabolism & Toxicology · doi:10.1517/17425255.2013.807798 · Cited by 120 · see citing works
“In vitro methods mentioned above [8-12,17] would be the candidates for first choice of method standardization.”
BackgroundMethodInfluentialThe role of gut microbial β-glucuronidase in drug disposition and development
Gao, S., Sun, R., Singh, R., So, S. Y., Chan, C. T. Y., Savidge, T., & Hu, M. (2022) · Drug Discovery Today · doi:10.1016/j.drudis.2022.07.001 · Cited by 92 · see citing works
Exploring Drug Metabolism by the Gut Microbiota: Modes of Metabolism and Experimental Approaches
Dhurjad, P., Dhavaliker, C., Gupta, K., & Sonti, R. (2021) · Drug Metabolism and Disposition · doi:10.1124/dmd.121.000669 · Cited by 60 · see citing works
“However, diet and physiological factors have affected fecalase and caecalase activity, leading to intraand inter-individual differences(Yeo et al., 2012).”
BackgroundAbo-Kadoum, M. A., Abouelela, M. E., Mousa, A. A. A., Abo-Dahab, N. F., Mosa, M. A., Helmy, Y. A., & Hassane, A. M. A. (2022) · Frontiers in Microbiology · doi:10.3389/fmicb.2022.1010332 · Cited by 54 · see citing works
“Enzymatic biosynthesis of resveratrol could be a promising process due to its independence from cell growth, conservative process conditions, and qualification, thus minimizing the inverse impacts facing resveratrol biosynthesis (Park et al., 2010; Yeo et al., 2012).”
BackgroundHow to Determine the Role of the Microbiome in Drug Disposition
Bisanz, J. E., Spanogiannopoulos, P., Pieper, L. M., Bustion, A. E., & Turnbaugh, P. J. (2018) · Drug Metabolism and Disposition · doi:10.1124/dmd.118.083402 · Cited by 48 · see citing works
Jeong, J.-J., Le, T. H. V., Lee, S., Eun, S.-H., Nguyen, M. D., Park, J. H., & Kim, D. (2015) · International Immunopharmacology · doi:10.1016/j.intimp.2015.07.025 · Cited by 46 · see citing works
Role of intestinal microflora in xenobiotic‐induced toxicity
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Jang, S., Kim, K., Han, M. J., & Kim, D. (2014) · Journal of Medicinal Food · doi:10.1089/jmf.2013.3073 · Cited by 40 · see citing works
Ophiopogon Polysaccharide Promotes the In Vitro Metabolism of Ophiopogonins by Human Gut Microbiota
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“, β-D-glucosidase > β-D-xylosidase > β-D-fucosidase > α-L-rhamnosidase), these results were consistent with previous studies, and the differences were probably caused by the composition of gut microbiota [19,39,40].”
ResultIn vitro genotoxicity assessment of functional ingredients: DHA, rutin and α-tocopherol
Sanz-Serrano, J., Vettorazzi, A., Muruzabal, D., de Ceráin, A. L., & Azqueta, A. (2021) · Food and Chemical Toxicology · doi:10.1016/j.fct.2021.112237 · Cited by 15 · see citing works
Ebuzoeme, C., Etim, I., Ikimi, A., Song, J., Du, T., Hu, M., Liang, D., & Gao, S. (2021) · Pharmaceutics · doi:10.3390/pharmaceutics13071043 · Cited by 8 · see citing works
Wang, X., Lyu, Y., Cheng, S. W., et al. (2025) · Journal of Pharmaceutical and Biomedical Analysis · doi:10.1016/j.jpba.2025.116717 · Cited by 7 · see citing works
Lee, M.-Y., Kim, Y. J., Jeon, W., et al. (2025) · Drug and Chemical Toxicology · doi:10.1080/01480545.2025.2495363 · Cited by 3 · see citing works
“…activation is essential for generating mutagenic effects (Rueff et al., 1990), whereas Yeo et al. argued that metabolic activation was not required (Yeo et al., 2012). therefore, further studies are needed to elucidate the functional roles of rutin in human health. chlorogenic acid, a minor…”
Microbiome: pharmacokinetics, pharmacodynamics and drug/xenobiotic interactions
Ojezele, M. O. (2020) · African Journal of Clinical and Experimental Microbiology · doi:10.4314/ajcem.v21i2.1 · Cited by 2 · see citing works
Gut bacterial O -demethylation in microbiome-dependent drug response
Chiang, C.-K., Dibello, B. S., Park, J., Rondinella, J., & Wu, Q. (2026) · Gut Microbes Reports · doi:10.1080/29933935.2026.2709263
“30,31 An analogous standardized microbial or enzymatic panel enriched for O -demethylation activity could be useful for confirming microbiome-mediated demethylation chemistry, comparing substrates across studies, and prioritizing candidates for more detailed mechanistic work.”
- Authors
- Yeo, H. K.
- Affiliations
- Kyung Hee University (KR)
- Corresponding author
- Yeo, Hee Kyung
- Venue
- Journal of Microbiology and Biotechnology
- Published
- 2012-06-28
- Volume
- 22
- Issue
- 6
- Pages
- 838-848
- ISSN
- 1017-7825 (ISSN-L)
- 1738-8872
- OpenAlex
- W1988751947
- Cited by
- 16
- References
- 0
- Citations by year
- 2026: 1 · 2025: 2 · 2022: 2 · 2021: 3 · 2020: 1 · 2019: 2 · 2018: 1 · 2015: 1 · 2014: 1 · 2013: 1 · 2012: 1
- FWCI
- 0.42
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Journal of Microbiology and Biotechnology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Toxicology › Bioactive Compounds and Antitumor Agents
- Health Sciences › Medicine › Biochemistry › Phytochemicals and Antioxidant Activities
- Keywords
- Ames test, mutagenicity testing, glycoside hydrolases, gut microbiota, rutin, quercetin, kaempferol, Salmonella typhimurium, S9 mix, food mutagens
- MeSH terms
- Biological Products, Feces, Glycoside Hydrolases, Glycosides, Humans, Human Experimentation, Korea, Mutagens, Mutation, Plant Extracts, Salmonella typhimurium, Sophora
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