Electronic Supplementary materials for Beneficial Microbes 20230038: Quantitative assessment of urinary equol levels, equol-producing bacteria, and the faecal microbiota in healthy Japanese individuals

Equol (4’,7-isoflavandiol) has attracted considerable attention for its potential efficacy in treating hormonal diseases. In this study we collected samples from healthy Japanese individuals (n = 91) to observe the relationship between the abundance of equol-producing bacteria in their faeces and the concentration of equol in their urine. Quantitative polymerase chain reaction (qPCR) targeting the dihydrodaidzein reductase gene (dhdr) was used to detect equol-producing bacteria. Equol producers, who were defined as individuals with >1000 nmol/l equol in their urine, exhibited 4-8 log10 copies of dhdr/g faeces of equol-producing bacteria. We assessed the accuracy of these findings by determining the rate of correspondence between possessing equol-producing bacteria and producing urinary equol. Of the 91 participants, 33 were found to be positive for both equol-producing bacteria and urinary equol, 52 were negative for both, one was positive for equol-producing bacteria and negative for urinary equol, and five were negative for equol-producing bacteria and positive for urinary equol. The sensitivity and specificity of the qPCR for detecting equol-producing bacteria were 86.8% and 98.1%, respectively. On the whole, the presence of equol-producing bacteria and urinary equol displayed 93.4% concordance, with a kappa coefficient of 0.862. No apparent correlation was observed between dhdr copy number in the faeces and urinary equol concentrations. Analysis of the faecal microbiota showed that alpha diversity indices (OTU, ACE, Chao1, Shannon) were significantly higher in equol producers. Specifically, the relative abundance of phylum Pseudomonadota was increased in non-equol producers, while abundance of genus Alistipes, Barnesiella, Butyricimonas, Odoribacter, and Ruminococcus, which produce short chain fatty acids and/or hydrogen, were only observed in equol producers. These results suggest that a certain amount of equol-producing bacteria must be present in the intestine to produce detectable levels of equol, and that equol productivity might be affected by other components of the microbiota.