**1. Introduction**

The existence of an inter-individual variability in response to diet and lifestyle interventions is widely accepted [1]. A complex interaction between diet, human genome, and the gu<sup>t</sup> microbiome occurs and can determine the effects of dietary bioactives [2]. The gu<sup>t</sup> microbiota is a critical component that can alter the absorption and metabolism of foods, and thus the final effects on human health. However, although a growing body of studies exists, the mechanisms underlying these processes are complex and not entirely understood. In this context, isoflavones-plant-derived polyphenols found at a relatively high concentration in soy and soy-derived products have been related to diverse health benefits such as the prevention of chronic diseases, including hormone dependent cancer, cardiovascular diseases, osteoporosis, and postmenopausal syndrome [3]. Although there is scientific evidence of the beneficial effects in counteracting symptoms like hot flushes and vasomotor reactions in menopausal women [4], the European Food Safety Authority (EFSA) has refuted health claims about the role of isoflavones in body functions [5]. The clinical effectiveness of ingested isoflavones might be due to their ability to be converted

Azcárate-Peril, M.A.; Tojo,

Guadamuro, L.;

**Citation:**

 R.; Mayo, B.; Delgado, S. Impact of Dietary Isoflavone Supplementation on the Fecal Microbiota and Its Metabolites in Postmenopausal Women. *Int. J. Environ. Res. Public Health* **2021**, *18*, 7939. https://doi.org/10.3390/ ijerph18157939

Academic Editors: Pablo Roman and Diana María Cardona Mena

Received: 20 June 2021 Accepted: 22 July 2021 Published: 27 July 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

into active metabolites like equol [6,7]. This metabolite is the isoflavone-derived compound with the strongest estrogenic activity and antioxidant capacity, and is generated by specific members of the gu<sup>t</sup> microbiota. Only some individuals harbor the microbiota required for the conversion into equol, resulting in different metabotypes: equol producers and non-producers [8]. Remarkably, compared with that in Asian populations (50–60%), the equol producer metabotype has a prevalence of 25–30% in Western populations [7].

Although the full range of intestinal bacteria involved in equol formation remains unknown [8,9], most of the equol-producing bacteria characterized so far are members of the family *Coriobacteriaceae* [10]. Additionally, the microorganisms responsible for equol production might differ across individuals [11–13].

Like other polyphenols, isoflavones are metabolized by components of the microbiota, and at the same time, they could also modulate the composition and/or activity of the intestinal microbial populations [14]. Analysis of intestinal microbiota modifications after isoflavone consumption could give clues as to the microorganisms involved in its metabolism. Some studies analyzed the effects of the isoflavone intake on the gu<sup>t</sup> microbiota [15–19]. However, additional studies applying high-throughput approaches are still needed to determine low abundance microorganisms, like those probably involved in equol production.

This study aimed to determine changes in the intestinal microbiota induced by a 1-month period of isoflavone consumption and to explore changes related to the equol status metabotype. With this aim, high-throughput amplicon sequencing of the bacterial 16S rRNA gene was performed on fecal samples taken before and after isoflavone consumption by eight menopausal women (three equol producers and five equol non-producers). In addition, metabolite analysis of feces was performed using gas chromatography for determination of possible shifts in fatty acid excretion.

#### **2. Materials and Methods**

## *2.1. Human Volunteers*

Ethical approval for this study was obtained from the Bioethics Subcommittee of the Spanish Research Council (Consejo Superior de Investigaciones Científicas or CSIC) and the Regional Ethics Committee for Clinical Research of the Health Service of Asturias (Servicio de Salud del Principado de Asturias) (approval number: 15/2011), in compliance with the Declaration of Helsinki. Fecal samples were provided, with written consent, by eight postmenopausal women recruited during a preceding study [18] at the Gynecology and Obstetrics Unit (in collaboration with the Gastroenterology Department) of Cabueñes Hospital (Gijón, Spain). The participants did not suffer from any infectious diseases or intestinal disorder. Additionally, they had not received antibiotics or any other medication for at least 6 months prior to the collection of samples. The women had been identified with an equol-producing metabotype (or not), based on the levels of urinary equol excretion [20]. For the present work, we selected three of the women (WC, WG, and WP) with an equolproducing phenotype (urine equol > 1000 nM as defined by Rowland et al. [21]) and five women (WE, WH, WF, WL, and WN) with a non-producing phenotype (excreted equol in urine ranging from 0 to 377 nM). Participants reported consuming a normo-type, Mediterranean diet and did not start following a vegetarian, vegan, or special diet during the intervention period. Supplementation consisted of a daily oral intake (80 mg/day) in the morning of a commercial dietary supplement (Fisiogen; Zambon, Bresso, Italy) rich in soy isoflavones (55–72% genistin/genistein, 28–45% other isoflavones) for one month.
