Equol producers and non-producers. Assessment of short-read shotgun sequencing and microbiome analysis of faecal samples to discriminate between equol producers and non-producers

Among the isoflavones and isoflavone-derived metabolites, equol, which in the human gut is synthesized from daidzein by minority bacterial populations, shows the strongest estrogenic and antioxidant activity. The beneficial effects on human health of isoflavone consumption might be partially or indeed totally attributable to this equol. Although some of the bacterial strains involved in its formation have been identified, the interplay between the composition and functionality of the gut microbiota and equol producer phenotype has hardly been studied. In this study, three analytical pipelines for the taxonomic and functional annotation of shotgun sequencing data were used in the search for similarities and differences in the faecal metagenome of equol producer (n=3) and non-producer (n=2) women, with special focus on equol-producing taxa and their equol-associated genes. The taxonomic profiles of the faecal samples differed significantly depending on the analytical method followed, although the microbial diversity detected by each tool was very similar at the phylum, genus and species levels. In addition, equol-producing taxa were detected in both equol producer and non-producer women, but no correlation between the abundance of equol-producing taxa and the equol producer/non-producer phenotype was found. Indeed, functional metagenomic analysis was unable to identify the pathways and genes involved in equol production, even in samples from equol producers. By aligning the reductase-encoding genes involved in equol production with the collected metagenomics data, only two reads mapping to daidzein- and tetrahydrodaidzein-reductase sequences were recognized in a sample from an equol producer. In conclusion, the taxonomic analysis of metagenomic data might not be suitable for detecting and quantifying equol-producing microbes in human faeces. Functional analysis of the data might provide an alternative. However, to detect the genetic makeup of the minority gut populations, more extensive sequencing than that contemplated in the present study might be required.

在异黄酮（isoflavones）及其衍生代谢物中，由少数肠道菌群从大豆苷元（daidzein）合成的雌马酚（equol）展现出最强的雌激素样活性与抗氧化活性。人群摄入异黄酮对健康产生的有益效应，可能部分乃至完全归因于该雌马酚。尽管现已鉴定出部分参与雌马酚合成的菌株，但肠道微生物组的组成、功能与雌马酚产生表型之间的相互作用，仍鲜有研究。本研究采用三种针对鸟枪测序（shotgun sequencing）数据的分类学与功能注释分析流程，对3名雌马酚产生者与2名非产生者女性的粪便宏基因组进行差异与相似性分析，重点关注产雌马酚微生物类群及其相关基因。粪便样本的分类学特征因所用分析方法的不同呈现显著差异，但各工具在门、属、种水平上检测到的微生物多样性却极为相近。此外，在雌马酚产生者与非产生者女性体内均检测到了产雌马酚微生物类群，但未发现产雌马酚类群的丰度与雌马酚产生/非产生表型之间存在关联。即便在雌马酚产生者的样本中，功能宏基因组分析也未能鉴定出参与雌马酚合成的代谢通路与相关基因。通过将参与雌马酚合成的还原酶编码基因与收集的宏基因组数据进行比对，仅在1名雌马酚产生者的样本中，识别到2条匹配大豆苷元还原酶与四氢大豆苷元（tetrahydrodaidzein）还原酶序列的测序读段（reads）。综上，宏基因组数据的分类学分析或许并不适用于人类粪便中产雌马酚微生物的检测与定量，而功能组学分析或可作为替代方案。不过，若要鉴定肠道内少数菌群的遗传组成，可能需要开展比本研究规模更大的测序工作。