Programa de Doctorado en Ingeniería Química, Ambiental y Bioalimentaria Metabolismo de isoflavonas y formación de equol por del tracto gastrointestinal humano.

Lucía Vázquez Iglesias Tesis Doctoral Oviedo, 2020

Programa de Doctorado en Ingeniería Química, Ambiental y Bioalimentaria

Metabolismo de isoflavonas y formación de equol por bacterias del tracto gastrointestinal humano

Lucía Vázquez Iglesias

Tesis Doctoral

Oviedo, 2020

Este trabajo ha sido realizado en el Instituto de Productos Lácteos de Asturias (IPLA-CSIC)

AUTORIZACIÓN PARA LA PRESENTACIÓN DE TESIS DOCTORAL

Año Académico: 2019/2020

1.- Datos personales del autor de la Tesis Apellidos: Nombre: Vázquez Iglesias Lucía DNI/Pasaporte/NIE: Teléfono: Correo electrónico: 71668563B 609562520 [email protected]

2.- Datos académicos Programa de Doctorado cursado: Programa en Ingeniería Química, Ambiental y Bioalimentaria Órgano responsable:

) Universidad de Oviedo 8

1 Departamento/Instituto en el que presenta la Tesis Doctoral: 0

2 Departamento de Ingeniería Química y Tecnología del Medio Ambiente . g

e Título definitivo de la Tesis R

( Español/Otro Idioma: Inglés:

9 Metabolismo de isoflavonas y formación de 0

0 equol por bacterias del tracto production by from the -

A gastrointestinal humano.

O Rama de conocimiento: V

- Ingeniería y Arquitectura T A

M 3.- Autorización del Director/es y Tutor de la tesis -

R D/Dª: Baltasar Mayo Pérez DNI/Pasaporte/NIE: 10820266-P

O Departamento/Instituto: F Departamento de Microbiología y Bioquímica de Productos Lácteos Instituto de Productos Lácteos de Asturias ( IPLA-CSIC)

D/Dª: Ana Belén Flórez García DNI/Pasaporte/NIE: 09432953-D Departamento/Instituto/Institución: Departamento de Microbiología y Bioquímica de Productos Lácteos Instituto de Productos Lácteos de Asturias ( IPLA-CSIC) Autorización del Tutor de la tesis D/Dª: Clara González de los Reyes-Gavilán DNI/Pasaporte/NIE: 10594470-A Departamento/Instituto: Departamento de Microbiología y Bioquímica de Productos Lácteos Instituto de Productos Lácteos de Asturias ( IPLA-CSIC) Autoriza la presentación de la tesis doctoral en cumplimiento de lo establecido en el Art. 32 del Reglamento de los Estudios de Doctorado, aprobado por el Consejo de Gobierno, en su sesión del día 20 de julio de 2018 (BOPA del 9 de agosto de 2018)

Villaviciosa, 27 Agosto del 2020 Director/es de la Tesis Tutor de la Tesis

Fdo.: Baltasar Mayo/ Ana Belén Flórez Fdo.: Clara G. de los Reyes-Gavilán

SR. PRESIDENTE DE LA COMISIÓN ACADÉMICA DEL PROGRAMA DE DOCTORADO EN ______

ÍNDICE INDEX ÍNDICE/INDEX

Lista de abreviaturas i Lista de figuras y tablas iii Resumen/ Summary iv

INTRODUCCIÓN /INTRODUCTION

1. ISOFLAVONAS Y EQUOL 1.1 Isoflavonas 1.1.1 Polifenoles y fitoestrógenos 1 1.1.2 Isoflavonas 2 1.1.3 Isoflavonas y soja 3 1.1.4 Metabolismo de isoflavonas 4 1.2 Equol 1.2.1 Características generales 5 1.2.2 Metabolismo de daidzeína y formación de equol 5 1.3 Isoflavonas, equol y salud 1.3.1 Mecanismos de acción 7 1.3.2 Efectos beneficiosos del equol en la salud 8 1.3.2.1 Menopausia 10 1.3.2.2 Sistema cardiovascular 10 1.3.2.3 Salud ósea 11 1.3.2.4 Cánceres hormonodependientes 12 1.3.2.5 Sistema nervioso central 12 1.3.2.6 Otros efectos beneficiosos 13

2. MICROBIOTA INTESTINAL HUMANA 2.1. Composición y enterotipos 13 2.2. Funciones de la microbiota 15 2.2.1. Función metabólica 16 2.2.2. Función inmunológica 17 2.2.3. Función protectora 17 2.3. Factores que influyen en la microbiota 18 2.3.1. Microbiota y dieta 18 2.3.2. Alimentación funcional, probióticos y prebióticos 19 2.4. Métodos de estudio de la microbiota intestinal 20 2.4.1. Modelos intestinales 21

3. MICROBIOTA Y EQUOL 3.1. Fenotipo productor de equol 23 3.1.1. Influencia de la dieta en el fenotipo productor de equol 24 3.2. Microorganismos productores de equol 25 3.3. Caracterización molecular de la formación de equol 28 3.4. Producción biotecnológica de equol 29 3.5. Equol y alimentos funcionales 30

OBJETIVOS/OBJECTIVES 31

TRABAJO EXPERIMENTAL /EXPERIMENTAL WORK CAPÍTULO 1 37 Desarrollo de métodos para identificar y cuantificar poblaciones intestinales involucradas en el metabolismo de las isoflavonas y la producción de equol.

CAPÍTULO 2 70 Estudio de las relaciones e interacciones entre isoflavonas, equol y poblaciones bacterianas intestinales.

CAPÍTULO 3 100 Caracterización de la producción de equol en muestras fecales y bacterias productoras con el fin de maximizar su síntesis endógena y biotecnológica.

DISCUSIÓN/ DISCUSSION 148

CONCLUSIONES/ CONCLUSIONS 162

BIBLIOGRAFÍA/BIBLIOGRAPHY 166

ANEXOS/ANNEXES . Anexo I.- Revisión bibliográfica sobre el equol y sus efectos en la salud humana 186 . Anexo II.- Informe sobre la calidad de los artículos 206

LISTA DE ABREVIATURAS

ADN: Ácido desoxirribonucleico FI: Factor de impacto AF: Alimentos funcionales FISH: “Fluorescence in situ hybridization” (Hibridación fluorescente in situ) AGCC: Ácidos grasos de cadena corta FOS: Fructooligosacáridos AOS: Arabinooligosacáridos g: Gramo ARN: Ácido ribonucleico GABA: “Gamma-aminobutyric acid” (Ácido ARNr: Ácido ribonucleico ribosomal gamma-aminobutírico) ATTC: “American Type Culture Collection” GOS: Galactooligosacáridos (Colección Americana de Cultivos Tipo) IgA: Inmunoglobulina A BAL: Bacterias ácido-lácticas ISAPP: “International Scientific Association for CCR: Cáncer colorrectal Probiotics and Prebiotics” (Asociación CIM: Concentración inhibitoria mínima Científica Internacional de Probióticos y cLDL: Colesterol unido a lipoproteínas de baja Prebióticos) densidad Kpb: Kilo pares de bases Ct: “Cycle threshold ” (ciclo umbral en qPCR) L: Litro DGGE: “Denaturing gradient gel electro- mL: Mililitro phoresis” (Electroforesis en geles de gradiente mg: Miligramos desnaturalizantes) MRSA: “Methicillin-resistant Staphylococcus DHD: Dihidrodaidzeína aureus” (S.aureus resistente a la meticilina) DSMZ: “Deutsche Sammlung von Mikroor- NADP(H): Nicotinamida adenina dinucleótido ganismen und Zellkulturen” (Colección fosfato Alemana de Microorganismos y Cultivos Celulares) NGS: “Next-generation sequencing” (Tecnologías de secuenciación masiva) ECV: Enfermedades cardiovasculares nmol: Nanomol EEUU: Estados Unidos de América O-DMA: O-desmetilangolensina EFSA: “European Food Safety Authority” (Autoridad Europea de Seguridad OMS: Organización Mundial de la Salud Alimentaria) ORFs: “Open reading frame” (Pauta abierta de EII: Enfermedad inflamatoria intestinal lectura) EPS: Exopolisacáridos PCR: “Polymerase chain reaction” (Reacción en cadena de la polimerasa) FAD: Flavín adenín dinucleótido Q: Cuartil FAO: “Food and Agriculture Organization of the United Nations” (Organización de las qPCR: “Quantitative polymerase chain Naciones Unidas para la Alimentación y la reaction” (PCR cuantitativa o PCR en tiempo Agricultura) real) FDA: “Food and Drug Administration” R-DHD: R-dihidrodaidzeína (Agencia Americana de Medicamentos y REs: Receptores estrogénicos Alimentación) REα: Receptor estrogénico subtipo alfa Fg: Femtogramo

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REβ: Receptor estrogénico subtipo beta Tm: “Melting temperature” (Temperatura de fusión) RT-PCR: “Reverse transcription polymerase chain reaction” (PCR con transcriptasa TNF α: “Tumor necrosis factor alpha” (Factor inversa) de necrosis tumoral alfa) RT-qPCR: “Quantitative reverse TNO: “Nederlandse Organisatie voor Toegepast transcription PCR” (PCR cuantitativa con Natuurwetenschappelijk Onderzoek” transcriptasa inversa) (Organización Holandesa para la Investigación Científica Aplicada) SCI: “ Science citation index” Ufc: Unidades formadoras de colonia S-DHD: S-dihidrodaizeína UHPLC: “Ultra-high-pressure liquid S-THD: S-tetrahidrodaizeína chromatography” (Cromatografía líquida de SHBG: “Sex hormone binding globulin” ultra-alta resolución) (Globulina fijadora de hormonas sexuales) UV: Ultravioleta TGI: Tracto gastrointestinal WHI: “Women’s Health Initiative” THD: Tetrahidrodaidzeína YIT: “Yeoju Institute of technology” (Instituto THS: Terapia hormonal sustitutiva de Tecnología de Yeoju)

TIM-2: “The TNO in vitro model of the colon” μ: Tasa específica de crecimiento (Modelo in vitro de colon desarrollado por el TNO) μg: Microgramo

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LISTA DE FIGURAS Y TABLAS

Figura/Tabla Título Página

Figura 1 Clasificación de los polifenoles según su estructura química: ácidos 1 fenólicos, lignanos, estilbenos y flavonoides, dentro de los cuales se incluyen las isoflavonas. Adaptado de Choi y Shin (2016).

Figura 2 Estructura química básica de las isoflavonas glicosiladas y agliconas. Se 3 indican los dos anillos fenólicos y el anillo cromano con las letras A, B y C, respectivamente, así como la numeración de los carbonos y la posición de los radicales cuyos sustituyentes se indican en la tabla adyacente.

Figura 3 Metabolismo de la daidzeína y ruta de formación de equol. Se indican las 6 enzimas de la microbiota intestinal humana involucradas en el metabolismo. Adaptado de Mayo et al. (2019).

Figura 4 Analogía estructural entre el 17 β-estradiol y el equol. 8

Figura 5 Efectos beneficiosos para la salud humana asociados al consumo de 9 equol.

Figura 6 Esquema de la distribución de la composición microbiana a lo largo del 14 tracto gastrointestinal humano en el que se representan los géneros más abundantes, el pH de la sección y la concentración bacteriana aproximada por gramos de contenido fecal. Adaptado de Jandhyala et al. (2015).

Figura 7 Esquema del modelo intestinal in vitro TIM-2. El sistema está integrado 22 por: (a) compartimentos con movimientos peristálticos que albergan la materia fecal y el medio de cultivo, (b) sensor de pH, (c) bombas de álcali, (d) sistema de diálisis con membrana semipermeable, (e) sensor

de nivel, (f) entrada de gas N2, (g) zona de muestreo, (h) salida de gas N2, (i) jeringuilla de alimentación y (j) sensor de temperatura. Imagen tomada de Rehman et al. (2012).

Figura 8 Árbol filogenético de la clase Coriobacteriia dentro del filo 27 adaptado de Nouioui et al. (2018). La primera rama incluye el órden Eggerthellales (verde claro) con la familia Eggerthellaceae (verde) y en la segunda rama el órden Coriobacteriales (naranja claro) integrado por las familias Coriobacteriaceae (amarillo) y Atopobiaceae (naranja). El superíndice “T” hace referencia a la cepa tipo. DSMZ: Colección alemana de microorganismos y cultivos celulares; ATTC: Colección americana de cultivos tipo, YIT: Instituto de tecnología de Yeoju.

Tabla 1 Bacterias involucradas en el metabolismo de daidzeína y del 25 intermediario dihidrodaidzeína (DHD) y la consecuente producción de los metabolitos derivados finales S-equol y O-desmetilangolensina (O- DMA).

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RESUMEN SUMMARY Resumen

RESUMEN

Las isoflavonas y sus metabolitos se asocian con diversos efectos beneficiosos para la salud humana. Tras su ingesta, se metabolizan por la acción de enzimas celulares y otros de la microbiota intestinal que las convierten en compuestos más biodisponibles, inactivos o más activos, como el equol a partir de la daidzeína. El equol es el metabolito de las isoflavonas con mayor poder estrogénico y capacidad antioxidante. Su biosíntesis tiene lugar por la acción secuencial de tres reductasas bacterianas. Sin embargo, el fenotipo productor de equol está presente solo en un 25-50% de la población. Los sujetos productores de equol pudieran ser los que más se benefician del consumo de isoflavonas. En la actualidad, no se sabe con exactitud qué componentes de la microbiota son responsables de la producción de equol, ni qué factores favorecen su desarrollo o actividad. En los microorganismos implicados, además, las rutas metabólicas, los enzimas responsables y los mecanismos de regulación son poco conocidos. Este conocimiento es esencial para extender los beneficios del consumo de isoflavonas a la población general con independencia de los taxones presentes en sus intestinos. En este contexto, en esta Tesis Doctoral se plantearon los siguientes objetivos: (i) desarrollar métodos para la detección y cuantificación de las poblaciones involucradas en el metabolismo de las isoflavonas y en la producción de equol, (ii) estudiar la interacción entre isoflavonas y poblaciones microbianas intestinales con especial interés en las que intervienen en la formación de equol y (iii) caracterizar la producción de equol en muestras fecales y en bacterias productoras con el fin de maximizar la formación endógena y su producción biotecnológica.

Para la detección y monitorización de genes involucrados en la síntesis de equol y los microorganismos responsables, se desarrollaron y aplicaron técnicas de qPCR y metagenómica no dirigida (shotgun). El método de qPCR permitió la monitorización de los genes de producción de equol, y en consecuencia las bacterias que los portan, en muestras fecales de mujeres productoras y no productoras. En este sentido, la secuenciación shotgun detectó unas pocas secuencias de taxones productores de equol, aunque la cantidad resultó similar en muestras de mujeres productoras y no productoras. La profundidad de secuenciación alcanzada, sin embargo, no permitió detectar genes involucrados en la síntesis de equol. El estudio de las interacciones isoflavonas-microbiota mostró que, aunque sin apenas efecto antimicrobiano, las isoflavonas parecen ser capaces de modular el desarrollo de diversas poblaciones intestinales, favoreciendo o inhibiendo su crecimiento de forma selectiva. Mediante el cultivo de aislados intestinales en medios

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Resumen con isoflavonas, se identificaron biotipos capaces de transformar las isoflavonas en compuestos derivados. Sin embargo, no identificamos bacterias capaces de producir equol, ni siquiera a pesar de que una de las cepas pertenecía a la especie productora Adlercreutzia equolifaciens. La secuenciación y análisis del genoma de esta cepa (IPLA 37004), y su comparación con el genoma de la cepa tipo (DSM19450T), reveló la deleción completa en la primera del clúster de síntesis de equol. La comparación genómica de esta y otras cepas de especies emparentadas mostró que la deleción de los genes de producción de equol está muy extendida, lo que sugiere que, en la actualidad, en el intestino humano el fenotipo productor no ofrece a las bacterias una ventaja selectiva. El tercer objetivo se abordó en tres trabajos independientes. Mediante la utilización de un modelo de intestino artificial, demostramos que una dieta rica en carbohidratos es capaz de duplicar la producción endógena de equol, sin incrementar, aparentemente, las poblaciones productoras. En el segundo, se estudió la transcripción de los genes del clúster de equol en A. equolifaciens DSM19450T. Este análisis reveló la presencia de un operón constituido por 13 genes contiguos que incrementan su expresión en presencia de daidzeína. En un artículo final, se sintetizó una secuencia de ADN con cuatro genes basada en el operón de esta cepa y se clonó en Escherichia coli y en Lactobacillus casei. Las cepas recombinantes de E. coli produjeron equol a partir de daidzeína mientras que los transformantes de L. casei solo produjeron equol con dihidrodaidzeína como sustrato.

Aunque son necesarios más estudios, los resultados de esta Tesis contribuyen a desentrañar las complejas relaciones isoflavonas-microbiota y sientan las bases para profundizar en mejorar la producción endógena y heteróloga de equol.

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Summary

SUMMARY

Isoflavones and their metabolites are endowed with various beneficial effects on human health. After intake, isoflavones are metabolized through the action of cellular enzymes and enzymes from the , which convert them into more bioavailable, inactive or more active compounds, such as equol from daidzein. Equol is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. Its biosynthesis proceeds via the sequential action of three bacterial reductases. However, the equol-producing phenotype is displayed by only 25-50% of the human population. These individuals could be the ones who benefit the most from isoflavone consumption. Currently, it is not yet known which components of the microbiota are responsible for equol production, nor what factors increase their number or their activity. Further, the metabolic pathways, the enzymes involved, and the regulatory mechanisms in equol- producing microorganisms are currently poorly understood. This knowledge is pivotal to extend the benefits of isoflavone consumption to the general human population regardless of the taxa present in their microbiota. In this context, in this Ph.D. Thesis, the following objectives were addressed: (i) to develop molecular methods for the detection and quantification of intestinal microbial populations involved in isoflavone metabolism and equol production, (ii) to study the interactions between isoflavones and intestinal microorganisms with a special interest in those producing equol, and (iii) to characterize equol production in faecal cultures and by equol-producing bacteria to maximize the endogenous formation and the biotechnological production of the compound.

For the detection and monitoring of genes involved in the synthesis of equol and its producing microbes, qPCR and untargeted metagenomics (shotgun) techniques were developed and applied. The qPCR method allowed the detection and quantification of equol-related genes, and as a consequence the bacteria that carry them, in faecal samples from equol-producing and non-producing women. In the same way, shotgun sequencing detected a few sequences of equol-producing taxa, although the numbers were similar in samples from equol-producer and non-producer women. The depth of sequencing attained, however, did not allow the detection of genes involved in equol synthesis. The study of the interactions between isoflavones and the microbiota showed that, although with little antimicrobial activity, isoflavones could be capable of modulating the development of various intestinal populations by favouring or inhibiting their selective growth. By culturing intestinal isolates in media with isoflavones, biotypes able to

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Summary transform isoflavones into isoflavone-derived compounds were identified. However, bacteria capable of producing equol were not detected, even though one of the strains belonged to the equol-producing species Adlercreutzia equolifaciens. Sequencing and analysis of the genome of this strain (IPLA 37004) and its comparison with the genome of the type strain (DSM19450T), revealed the complete deletion of the equol synthesis cluster in the former. Genome comparison of that and other strains of related species showed that the deletion of equol-producing genes is widespread, suggesting that the producing phenotype does not currently offer a selective advantage in the human intestine to the producing bacteria. The third objective was addressed in three independent works. By using an artificial model of the human intestine, we demonstrated that a diet rich in carbohydrates was capable of doubling the production of endogenous equol without an apparent increase of the producing populations. In the second one, we studied the transcription of genes from the equol cluster of A. equolifaciens DSM19450T. This analysis revealed the presence of an operon made up of 13 contiguous genes that increased their expression in the presence of daidzein. In a last work, a DNA sequence based on the operon of this strain was synthesized and cloned in Escherichia coli and Lactobacillus casei. The recombinant strains of E. coli produced equol from daidzein, while L. casei transformants of produced equol only when dihydrodaidzein is used as a substrate.

Although more studies will be required, the results of this Thesis contribute to unravelling the complex isoflavone-microbiota relationships and lay the foundations for fostering an increase of endogenous equol formation and its heterologous production.

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INTRODUCCIÓN INTRODUCTION

Introducción

1. ISOFLAVONAS Y EQUOL

1.1. ISOFLAVONAS

1.1.1. Polifenoles y fitoestrógenos

Los polifenoles constituyen uno de los grupos de compuestos químicos más numerosos y ampliamente distribuidos en el reino vegetal (Tsao, 2010). Hay descritos más de 10.000 compuestos polifenólicos distintos cuya estructura molecular básica se caracteriza por presentar al menos un anillo aromático (fenólico) unido a uno o más grupos hidroxilo (Crozier et al., 2009; Li et al., 2014). La gran diversidad y amplia distribución de estos compuestos naturales en las plantas permite clasificarlos en función de su origen, actividad biológica o estructura química. Así, atendiendo al número de anillos fenólicos que contienen y a los elementos estructurales que unen dichos anillos, los polifenoles se subdividen en: (a) ácidos fenólicos (p. ej., ácido gálico y ácido cumárico), (b) flavonoides (p. ej., cuercitina y daidzeína), (c) estilbenos (p. ej., resveratrol) y (d) lignanos (p. ej., secoisolariciresinol) (Manach et al., 2004) (Figura 1).

Cianidina

Hesperetina Catequina Apigenina Cuercitina

DAIDZEÍNA

GENISTEÍNA GLICITEÍNA

Figura 1.- Clasificación de los polifenoles según su estructura química: ácidos fenólicos, lignanos, estilbenos y flavonoides, dentro de los cuales se incluyen las isoflavonas. Adaptado de Choi y Shin (2016).

Los polifenoles son compuestos del metabolismo secundario de las plantas en las que ejercen diversas funciones: protección frente a las infecciones bacterianas, atrayentes de polinizadores y animales, protectores de la radiación UV, moléculas de señalización en la

1 Introducción formación de nódulos en la raíz por los fijadores de nitrógeno, etc. (Del Rio et al., 2013). Además, el consumo de polifenoles se ha relacionado abundantemente con efectos beneficiosos sobre la salud humana, disminuyendo, por ejemplo, los síntomas de la menopausia, o ejerciendo una protección frente a enfermedades crónicas como la osteoporosis, las enfermedades cardiovasculares y ciertos tipos de cáncer (Landete et al., 2016). Los fitoestrógenos son un grupo heterogéneo de compuestos polifenólicos que poseen una gran similitud estructural con la hormona sexual femenina estradiol (17-β-estradiol), lo que les confiere propiedades similares a los estrógenos (Pilsaková et al., 2010). Existen cinco grandes familias de compuestos polifenólicos con actividad estrogénica: isoflavonas, estilbenos, lignanos, elagitaninos y cumestanos (Landete et al., 2016); de ellos los más estudiados, sin duda, son las isoflavonas. Como otros polifenoles, los fitoestrógenos se encuentran en las plantas en forma de glicósidos unidos a residuos de azúcar. Para que alcancen su máxima biodisponibilidad y actividad estrogénica es necesario escindir estos residuos azucarados, lo que resulta en la liberación de sus agliconas que, en general, son más activas que las formas glicosiladas (Kolátorová et al., 2018).

1.1.2. Isoflavonas

Las isoflavonas son el grupo más destacado y estudiado de los fitoestrógenos debido a los beneficios que parecen aportar a la salud humana (Durazzo et al., 2019). Químicamente, se caracterizan por poseer como estructura básica un esqueleto C6-C3-C6, en los que las dos unidades C6 (los anillos A y B) tienen naturaleza fenólica mientras que la unidad C3 es un anillo cromano (anillo C). La presencia de distintos radicales en diversas posiciones da lugar a un gran número de compuestos distintos. De esta forma, la diferencia entre los principales tipos de isoflavonas radica en los sustituyentes de las posiciones 5, 6 y 7 del anillo A (radicales R1, R2 y R3, respectivamente) (Figura 2). Las isoflavonas de la soja se encuentran en las plantas unidas a residuos de glucosa en la posición 7 del anillo A (Chandrasekharan y Aglin, 2013), dando lugar a las formas glicosiladas daidzina, genistina y glicitina. Estos glucósidos, a su vez, es muy frecuente que se encuentren unidos a formas acetil, malonil o succinil, dando lugar a los correspondientes acetilglucósidos, malonilglucósidos o succinilgluclósidos (Křížová et al., 2019).

2 Introducción

R1 R2 R3 8 AGLICONAS R3 9 2 Daidzeína -H -H -OH 7 A Genisteína -OH -H -OH C 2´ 3 4 Gliciteína -H -OCH3 -OH 6 1´ 3´ R2 5 GLUCÓSIDOS

B Daidzina -H -H Glucosa R1 6´ Genistina -OH -H Glucosa 4´ H

5´ Glicitina -H -OCH3 Glucosa

Figura 2.- Estructura química básica de las isoflavonas glicosiladas y agliconas. Se indican los dos anillos fenólicos y el anillo cromano con las letras A, B y C, respectivamente, así como la numeración de los carbonos y la posición de los radicales cuyos sustituyentes se indican en la tabla adyacente.

1.1.3. Isoflavonas y soja

Las isoflavonas están presentes en más de 300 tipos de plantas, principalmente en sus raíces y semillas (Klejdus et al., 2005). Las concentraciones más elevadas de isoflavonas se encuentran en diversas especies de la familia Fabaceae (Tsao, 2010) como la soja, los garbanzos, los cacahuetes, los guisantes o la alfalfa (Chandrasekharan y Aglin, 2013). De todas ellas, la soja (Glycine max), con un contenido medio de 1-2 mg/g, destaca como la fuente principal de isoflavonas en la dieta humana (Coward et al., 1993). La soja posee una composición nutricional caracterizada por un contenido elevado en proteínas con gran valor biológico y un bajo contenido en carbohidratos, fundamentalmente oligosacáridos; además, constituye una fuente importante de ácidos grasos esenciales y de una amplia variedad de vitaminas y minerales, entre los que destaca el potasio (Messina, 2016). Los beneficios del consumo de soja sobre la salud humana se han relacionado tradicionalmente con su contenido en isoflavonas. Sin embargo, en la soja existen también otros compuestos bioactivos como saponinas, fitoesterol, lignanos o ácido fítico que, bien solos o en combinación con las isoflavonas o las proteínas, podrían a su vez estar involucrados en estos efectos saludables (Kang et al., 2010). El consumo de soja, y en consecuencia de isoflavonas, varía considerablemente entre países orientales y occidentales, debido fundamentalmente al empleo de soja en la cocina tradicional asiática. Así, en Japón el consumo medio en adultos se sitúa entre 30 y 50 mg al día mientras que en Estados Unidos, Canadá o Europa no llega a 3 mg al día (Messina, 2016). La soja se consume a través de una amplia variedad de productos, incluyendo las alubias de la soja, harinas de soja, proteínas de soja aisladas o texturizadas y productos de soja fermentados como miso, tempeh, natto o salsa de soja (Chandrasekharan y Aglin, 2013). La concentración y las formas en las que las isoflavonas se encuentran en los

3 Introducción productos finales dependen en gran medida de la forma de elaboración y el procesado de los productos. En los alimentos crudos como la leche de soja, la mayor parte de las isoflavonas se presentan en sus formas glicosiladas (Barnes, 2010). Sin embargo, en los productos fermentados, la cantidad de agliconas es mayor debido a la hidrolisis de las formas glicosiladas durante la fermentación. Además de las que se ingieren con la dieta, las isoflavonas se pueden consumir a partir de numerosos suplementos alimenticios comerciales a base de extractos de soja enriquecidos en estos compuestos. Según una reciente evaluación de la Autoridad Europea de Seguridad Alimentaria, una ingesta diaria de isoflavonas de entre 20 y 150 mg/día se considera segura (EFSA ANS Panel, 2015).

1.1.4. Metabolismo de isoflavonas

La biodisponibilidad de los polifenoles de la dieta, y por tanto de las isoflavonas, se ve afectada por numerosos factores extrínsecos, como la variación del contenido de alimentos, la matriz o el procesado al que estos se han sometido, y también por factores intrínsecos, como el fondo genético de cada individuo o la composición de su microbiota intestinal (Kemperman et al., 2010). El metabolismo de los glucósidos de isoflavona comienza tras la ingestión del alimento cuando estos se liberan de la matriz vegetal y quedan accesibles para su desglicosilación. Esta reacción, la llevan a cabo enzimas tisulares y enzimas microbianas. Las formas conjugadas son probablemente demasiado hidrofílicas para atravesar por difusión pasiva la pared intestinal (Manach et al., 2004). Sin embargo, las formas desglicosiladas o agliconas (daidzeína, genisteína y gliciteína) tienen mayor hidrofobicidad y menor peso molecular, por lo que se cree que son estas formas las que mejor y más rápido se absorben (Izumi et al., 2000). La hidrólisis de las formas conjugadas es extremadamente eficiente y tiene lugar en las células del epitelio intestinal gracias a la acción de β-glucosidasas tisulares (Lampe, 2009). Una vez hidrolizadas, las agliconas se absorben por difusión pasiva y rápidamente se transforman o metabolizan en el epitelio intestinal o en el hígado. El metabolismo de las agliconas se lleva a cabo mayoritariamente por enzimas de la fase II (que unen las isoflavonas covalentemente a moléculas endógenas como ácido glucurónico, glutatión, sulfato o aminoácidos generando conjugados) y en menor medida por enzimas de fase I (que participan en fenómenos de oxidación, oxigenación, reducción o hidrólisis) (Lampe, 2009). Las isoflavonas conjugadas se secretan con la bilis y se liberan a la parte superior del intestino delgado donde se produce de nuevo una desconjugación microbiana; una parte de ellas se reabsorbe de nuevo y vuelven otra vez al hígado en un

4 Introducción proceso cíclico que se conoce como circulación enterohepática (Chandrasekharan y Aglin, 2013). Las isoflavonas que no son hidrolizadas y absorbidas en el intestino delgado junto con algunas de las excretadas se metabolizan en el colon por enzimas de la microbiota intestinal mediante reacciones de deshidroxilación, reducción, ruptura del anillo C o desmetilación (Setchell et al.,2002). En el colon se transforman en compuestos más activos que sus precursores, como es el caso del equol a partir de la daidzeína o en componentes inactivos (Setchell et al., 2005; Choi y Kim, 2014). La principal ruta de excreción de las isoflavonas es a través de la orina, aunque el porcentaje de las fracciones y los metabolitos recuperados son muy variable entre individuos. Así, los valores de recuperación se sitúan entre el 18 y el 95% para la daidzeína y entre el 5 y el 42% para la genisteína (Setchell et al., 2003a).

1.2. EQUOL

1.2.1. Características generales

El equol se sintetiza a partir de la daidzeína y se caracteriza por ser el metabolito derivado de las isoflavonas con mayor actividad estrogénica (Setchell et al., 2002; Yuan et al., 2007) y mayor capacidad antioxidante (Sánchez-Calvo et al., 2013, Choi y Kim, 2014).

El equol posee la fórmula química C15H12O(OH)2 y se aisló e identificó por primera vez en 1932 a partir de la orina de yeguas preñadas (Marrian y Haslewood, 1932). Muchos años más tarde, en 1982, se convirtió en el primer compuesto derivado de las isoflavonas en ser detectado en orina y sangre humanas (Setchell y Clerici, 2010a). El equol se describe como un compuesto no polar, relativamente insoluble en solución acuosa y extremadamente sensible al ácido (Setchell y Clerici, 2010a). A diferencia de otras isoflavonas y metabolitos, el equol posee una estructura no planar, la cual es responsable, al menos en parte, de sus actividades fisiológicas (Crozier et al., 2009). Es un compuesto ópticamente activo debido a la presencia de un carbono asimétrico en la posición C3 que hace posible que existan dos formas enantiómeras: el S-(-)equol y el R- (+)equol (Setchell y Clerici, 2010a).

1.2.2. Metabolismo de daidzeína y formación de equol

En el colon la daidzeína se transforma en el intermediario dihidrodaidzeína (DHD) que más adelante se metaboliza en O-desmetilangolensina (O-DMA) o en equol (Clavel y Mapesa, 2013). Que se forme uno u otro compuesto viene determinado por los componentes de la microbiota intestinal de los individuos. Estos pueden ser capaces de

5 Introducción producir O-DMA, vía 2´-dehidro-O-desmetilangolensina, o equol, vía DHD y tetrahidrodaidzeína (THD) (Del Rio et al., 2013) (Figura 3).

Daidzina

Β-glucosidasas

Daidzeína reductasa

Daidzeína R(-)-dihidrodaidzeína (R-DHD) Dihidrodaidzeína racemasa

Dihidrodaidzeína reductasa

S(-)-dihidrodaidzeína S(-)-tetrahidrodaidzeína (S-DHD) (S-THD) ?? Tetrahidrodaidzeína reductasa

S(-)-equol 2´-dehidro-O-desmetilangolensina ??

O-desmetilangolensina (O-DMA)

Figura 3.- Metabolismo de la daidzeína y ruta de formación de equol. Se indican las enzimas de la microbiota intestinal humana involucradas en el metabolismo. Adaptado de Mayo et al. (2019).

El O-DMA es un metabolito sin aparente actividad estrogénica y lo produce entre un 80 y un 90% de los individuos (Frankenfeld, 2011b). El equol, por el contrario, es un metabolito más activo que su precursor, pero solo lo producen entre el 25 y el 50% de los sujetos (Setchell y Cole, 2006). La formación de equol depende de la composición de la microbiota intestinal, ya que los animales libres de gérmenes son incapaces de producir este compuesto (Axelson y Setchell, 1981). Las bacterias intestinales sintetizan únicamente la forma enantiomérica S-(-)equol (Setchell et al., 2005); una vez producido, el compuesto parece ser bastante estable (Setchell et al., 2002).

6 Introducción

Además de la síntesis endógena, el equol puede ser ingerido como suplemento alimenticio. Tras el consumo, este compuesto se absorbe rápidamente y muestra una máxima concentración en plasma aproximadamente a las seis horas; en orina se detecta hasta 12 horas tras su ingesta (Setchell et al., 2009).

1.3. ISOFLAVONAS, EQUOL Y SALUD

1.3.1. Mecanismos de acción

Debido a la similitud estructural con el 17-β-estradiol, las isoflavonas actúan interaccionado con los receptores estrogénicos (REs) (Barnes et al., 2000) y modulando la unión del estradiol (Yuan et al., 2007) (Figura 4). Los REs pertenecen a la superfamilia de receptores intracelulares de tipo esteroideo/tiroideo y se localizan principalmente en la membrana del núcleo (Pilsaková et al., 2010). Estos receptores se presentan en dos formas: subtipo alfa (REα) y subtipo beta (REβ), los cuales presentan una expresión diferencial en distintos tejidos. El subtipo REα tiene más representación en las glándulas mamarias y el útero, participando en la homeostasis del esqueleto y la regulación del metabolismo, mientras que el subtipo REβ se expresa predominantemente en el colon, médula osea y epitelio glandular y tiene un mayor efecto sobre el sistema nervioso central y el sistema inmune (Paterni et al., 2014). En general, la afinidad de las isoflavonas por los REs es aproximadamente 100-500 veces menor que la que posee el 17-β-estradiol (Pilsaková et al., 2010). Además, la afinidad por los REα y REβ de las distintas isoflavonas es a su vez desigual. Así, la afinidad de la genisteína por los REβ es unas 20-30 veces mayor que por los REα (Kuiper et al., 1998). Los receptores REα y REβ actúan también como factores de transcripción nucleares, de forma que se asocian con los elementos de respuesta estrogénicos y se unen a determinadas secuencias reguladoras del ADN activando la transcripción de los genes diana (Paterni et al., 2014). Los receptores desencadenan también efectos a través de la activación de receptores de membrana que producen una cascada de respuestas entre las que se incluyen el control de la actividad de las proteínas G, la adenilatociclasa, la fosfolipasa o las proteín kinasas (Simoncini et al., 2003). Otra posible vía de actuación de las isoflavonas en el organismo es a través de la interacción con las hormonas sexuales esteroideas inhibiendo la actividad de la 5α-reductasa (que cataliza la transformación de testosterona en 5α-dihidrotestosterona) y la aromatasa P450 (que interviene en la conversión de testosterona en estradiol). Finalmente, las isoflavonas pueden unirse a la globulina fijadora de hormonas sexuales (SHBG) estimulando su síntesis y provocando cambios en la concentración de las hormonas circulantes (Pilsaková et al., 2010).

7 Introducción

Como el de las isoflavonas, el principal mecanismo de actuación del equol está mediado por su unión a los REs de las células. La afinidad del equol por estos receptores es mayor que la que posee su precursor la daidzeína, dando lugar, por tanto, a una mayor actividad estrogénica (Lehmann et al., 2005). El isómero natural S-(-)equol exhibe una fuerte afinidad de unión por los Reβ (Muthyala et al., 2004). Además de las propiedades estrogénicas, el equol tiene también propiedades antiandrogénicas, ya que es capaz de unirse específicamente a la 5α-dihidrotestosterona inhibiendo la unión de esta molécula a sus receptores alterando, por tanto, la respuesta hormonal (Lund et al., 2004). Esto pudiera tener particular importancia en la prevención y tratamiento del cáncer de próstata. Finalmente, el equol, como compuesto polifenólico que es, podría actuar a través de su potente actividad antioxidante. La capacidad antioxidante ha sido evaluada en estudios in vivo e in vitro donde se ha visto que tienen mayor actividad antioxidante que la daidzeína y las demás isoflavonas y sus metabolitos (Sánchez-Calvo et al., 2013). La actividad antioxidante se debe, posiblemente, al alto número de electrones libres de su molécula, lo que lo convierte en un buen donador de hidrógenos con capacidad de eliminar los radicales libres (Setchell y Clerici, 2010b). El equol pudiera actuar también induciendo la expresión de enzimas involucradas en la defensa frente al estrés oxidativo como la superóxido dismutasa, la catalasa y la glutatión peroxidasa (Alfa y Arroo, 2019).

17β-estradiol CH

Equol

Figura 4.- Analogía estructural entre el 17 β-estradiol y el equol.

1.3.2. Efectos beneficiosos del equol en la salud

El consumo de isoflavonas se ha relacionado tradicionalmente con diversos efectos beneficiosos para la salud humana (Durazzo et al., 2019). Uno de los efectos más estudiado es el tratamiendo de los síntomas relacionados con la menopausia. En comparación con las poblaciones occidentales, las mujeres asiáticas sufren menos sofocos, sudoraciones nocturnas y otros síntomas de la menopausia, lo que se ha asociado con el elevado consumo de soja y sus derivados típicos en la dieta asiática (Messina, 2000). El consumo de soja se relaciona también con un efecto protector frente a enfermedades

8 Introducción cardiovasculares (Pilsaková et al., 2010), una reducción de la resorción de hueso característica de la osteoporosis en la mujer (Wei et al., 2012), una menor incidencia de cánceres hormonodependientes como el cáncer de mama (Qiu et al., 2019) o de próstata (Mahmoud et al., 2014) y una limitada mejora de la función cognitiva (Soni et al., 2014). Sin embargo, a pesar de las evidencias epidemiológicas, los resultados obtenidos en los estudios de intervención controlados y en los meta-análisis subsiguientes no son todavía concluyentes (Bolaños et al., 2010). Dado que el equol es el metabolito derivado de las isoflavonas con mayor capacidad estrogénica y antioxidante, este compuesto podría ocupar un papel destacado en los efectos beneficiosos sobre la salud humana asociados a la ingesta de soja (Mayo et al., 2019). Esto explicaría, al menos en parte, los resultados discordantes obtenidos en las intervenciones con isoflavonas, ya que sólo una pequeña porción de las poblaciones es capaz de producir este compuesto. Por este motivo, los resultados de las intervenciones con isoflavonas de soja se tienden a evaluar en la actualidad en función del fenotipo productor o no productor de equol (Ishimi, 2010; Weaver y Legette, 2010; Hazim et al., 2016; Igase et al., 2017). A pesar de la dificultad de obtención de cantidades adecuadas de equol para su evaluación experimental, en los últimos años se han llevado a cabo numerosas intervenciones experimentales con el compuesto puro para evaluar la prevención o el alivio de diversas enfermedades y síndromes (Tousen et al. 2011b; Aso et al., 2012; Usui et al., 2013; Daily et al., 2018; Yoshikata et al., 2018). A modo de resumen, en la Figura 5 puede verse un diagrama de los sistemas del organismo sobre los que el equol actúa ejerciendo un efecto beneficioso para la salud.

Figura 5.- Efectos beneficiosos para la salud humana asociados al consumo de equol.

9 Introducción

1.3.2.1. Menopausia

Durante décadas la terapia hormonal sustitutiva (THS) se consideró el tratamiento de elección para mitigar los síntomas de la menopausia. Sin embargo, la publicación de los resultados de la “Women’s Health Initiative” (WHI) en el año 2002, en la que se sugería una mayor incidencia de cáncer de mama, útero y enfermedades cardiovasculares en mujeres tratadas con THS, conllevó un descenso acusado del uso de la THS y, en consecuencia, la búsqueda de terapias alternativas (Utian et al., 2015). En la actualidad, las conclusiones obtenidas en el estudio del WHI están siendo sometidas a revisión y la asociación entre THS y enfermedades no parece tan concluyente (Lobo, 2016; Langer, 2017). Debido a su similitud química con el estradiol, las isoflavonas estuvieron entre las primeras moléculas analizadas para reemplazar la THS (Messina, 2000; Bolaños et al., 2010). Dada su mayor actividad estrogénica, el equol podría ser una de las isoflavonas con un papel más destacado en la disminución de los síntomas de la menopausia. Los resultados obtenidos hasta la fecha, sin embargo, son contradictorios y poco concluyentes, aunque hay muchos estudios de intervención en los que el equol parece mejorar la sintomatología de la menopausia. Así, un suplemento de 10 mg de S-equol al día resultaba efectivo para aliviar la rigidez de los músculos y el dolor de las articulaciones en mujeres menopáusicas (Aso et al., 2012; Jenks et al., 2012), mejorando ocasionalmente el estado de ánimo (Ishiwata et al., 2009). Otros autores han descrito que una dosis igual o superior a 20 mg de S-equol disminuía significativamente los sofocos (Jenks et al., 2012; Daily et al., 2018). Finalmente, estudios recientes de meta-análisis de numerosos ensayos revelan que el equol disminuía significativamente la incidencia y/o severidad de los sofocos de la menopausia (Daily et al., 2018).

1.3.2.2. Sistema cardiovascular

Las enfermedades cardiovasculares (ECV), entre las que se incluyen las enfermedades coronarias y los accidentes cerebrovasculares, son la principal causa de muerte en el mundo (Lou et al., 2016). Las altas tasas de mortalidad por ECV se deben en gran medida a factores genéticos, ambientales, culturales o a diferencias en el estilo de vida como la actividad física, el alcohol, el tabaco y la dieta (Doughty et al., 2017). Estos factores presentan una gran variabilidad entre los individuos de diferentes poblaciones. Diversos estudios observacionales llevados a cabo en regiones con alta tradición en el consumo de soja muestran una menor incidencia de enfermedades coronarias (Messina, 2010). El equol se ha relacionado también con la prevención de ECV mediante estudios observacionales e intervencionales. El efecto preventivo podría resultar de la inhibición de

10 Introducción la actividad aterogénica (potencialidad de obstrucción de las arterias) y la capacidad de mejorar la elasticidad arterial que posee este metabolito (Sekikawa et al., 2018). Estudios de intervención en los que se suministró equol a un grupo de hombres y mujeres con sobrepeso u obesidad, concluyeron con una disminución significativa del riesgo de ECV cuantificado mediante la reducción de los niveles serológicos del colesterol unido a las lipoproteínas de baja densidad (cLDL) y del índice vascular corazón-tobillo que evalúa la rigidez arterial (Usui et al., 2013). El efecto más destacado del equol se observó en el subgrupo de mujeres que no eran capaces de producirlo. En otro estudio, la administración de un suplemento oral de equol durante un año a mujeres de mediana edad también mostró una mejora en parámetros cardiovasculares (Yoshikata et al., 2018).

1.3.2.3. Salud ósea

Asociado al descenso acusado de los niveles de estrógenos característico de la menopausia, se produce un aumento de la tasa de remodelación ósea (menor formación y mayor resorción) y, por consiguiente, disminuye la densidad mineral ósea, lo que conduce a la osteoporosis (Wei et al., 2012). Con mayor o menor evidencia se le atribuyen a las isoflavonas de la soja efectos en la prevención de la osteoporosis en mujeres menopáusicas (Cassidy et al., 2006; Wei et al., 2012; Pawlowski et al., 2015). No obstante, existen otros estudios en los que el consumo de isoflavonas no mostró ningún efecto beneficioso en ese sentido (Tai et al., 2012). El equol parece poseer también propiedades inhibitorias en el proceso de resorción ósea, caracterizado por una eliminación de tejido óseo por los osteoclastos. Esta inhibición del equol se conseguiría por una reducción de la formación de osteoclastos (Ohtomo et al., 2008; Tousen et al., 2011b), a través de la inhibición de la expresión del factor de necrosis tumoral alfa (TNF α) que estimula la formación osteoclástica (Kang et al., 2005). El equol parece reducir también la expresión de genes asociados con la inhibición de la formación ósea y la destrucción de cartílago (Lin et al., 2016). El efecto de la preservación ósea tras el consumo de equol se ha observado en ratas con bajos niveles de estrógenos y en ratones ovariectomizados (Fujioka et al., 2004; Rachoń et al., 2007). En este mismo sentido, varios estudios han mostrado que un suplemento natural de S-equol de 10 mg al día durante 1 año contribuía al mantenimiento de la salud ósea de mujeres, inhibiendo la reabsorción y disminuyendo la pérdida de densidad del hueso (Tousen et al., 2011b; Yoshikata et al., 2018).

11 Introducción

1.3.2.4. Cánceres hormonodependientes

En países orientales, la incidencia de cáncer de próstata, colon y algunos cánceres de mama es mucho menor en comparación con la de países occidentales (Fritz et al., 2013; Bilal et al., 2014; Messina, 2016). Esta baja incidencia aumenta en pocas generaciones cuando las poblaciones asiáticas emigran a países occidentales y abandonan sus hábitos dietéticos ancestrales (Ziegler et al., 1993; Stanford et al., 1995). En relación con el cáncer, hay pocos trabajos observacionales con equol, y la mayoría se han realizado en individuos con fenotipo productor. En algunos trabajos se observa una tendencia entre los niveles de equol en el plasma y una reducción del riesgo de desarrollar cáncer de mama, aunque las diferencias no son significativas (Verheus et al., 2007). Por el contrario, otros estudios muestran que las concentraciones de equol en plasma resultaban similares en mujeres con y sin cáncer de mama, señalando una ausencia de asociación del equol con este tipo de cáncer (Wu et al., 2004). Incluso, en otros trabajos, se han descrito correlaciones positivas entre el equol y la incidencia de cáncer de mama (Grace et al. 2004). En hombres, se ha sugerido que el equol podría jugar un papel importante en la prevención del cáncer de próstata (Takahashi et al., 2006), ya que, en diversas poblaciones asiáticas, la producción de equol se relaciona con una disminución del riesgo de este tipo de cáncer (Akaza et al., 2004; Mahmoud et al., 2014).

1.3.2.5. Sistema nervioso central

Debido fundamentalmente al envejecimiento de la población, se calcula que para el año 2050 el número de personas afectadas por demencia en todo el mundo se aproximará a los 130 millones (Wolters y Ikram, 2018). Varios estudios epidemiológicos señalan que las tasas de demencia son inferiores en las poblaciones asiáticas con respecto a las de países occidentales (Liu et al., 2003). Las isoflavonas de la soja podrían tener un papel en la prevención del déficit cognitivo, aspecto que ha sido ampliamente estudiado, aunque con resultados poco concluyentes. Aproximadamente la mitad de los estudios observacionales y de las pruebas aleatorias controladas realizadas en humanos señalan efectos beneficiosos de las isoflavonas sobre la función cognitiva (Soni et al., 2014). Una revisión de los estudios clínicos y epidemiológicos sugirió que las isoflavonas de la soja mejoraban la función cognitiva en mujeres, pero los resultados en hombres resultaron más inconsistentes (Lee et al., 2005). Las propiedades antiaterogénicas del S-equol podrían contribuir a disminuir la ateroesclerosis y la rigidez arterial lo que podría tener un efecto beneficioso en la prevención de la demencia y del déficit cognitivo (Igase et al., 2017; Sekikawa et al., 2018).

12 Introducción

1.3.2.6. Otros efectos beneficiosos

En mayor medida que las isoflavonas, el equol parece ayudar a mejorar la salud de la piel y a luchar contra los signos de la edad (Magnet et al., 2017). Esta actividad podría deberse a su gran capacidad antioxidante, fitoestrogénica y, posiblemente, a efectos epigenéticos. La suplementación de la dieta de mujeres menopáusicas con 10-30 mg de S- equol al día durante 12 meses redujo la profundidad de los pliegues de la piel y el área total de las arrugas (Oyama et al., 2012). El equol parece tener también un efecto sobre aspectos de la superficie de la piel como la rugosidad, textura, suavidad, elasticidad y firmeza, así como sobre algunos parámetros moleculares que influyen en su mantenimiento (longitud de los telómeros y la metilación LINE-1 del ADN) (Magnet et al., 2017). Además de en la piel, algunos autores sugieren que, a través del control glicémico, el equol, pero no las isoflavonas, pudiera tener también un papel importante en la prevención de la diabetes de tipo 2 (Usui et al., 2013; Dong et al. , 2020).

2. MICROBIOTA INTESTINAL HUMANA

2.1. Composición y enterotipos

El tracto gastrointestinal (TGI) humano alberga un gran número de diversos microorganismos que en su conjunto se conocen con el término de microbiota intestinal. La microbiota está compuesta por bacterias, arqueas, hongos (mohos y levaduras), protozoos y virus (principalmente bacteriófagos). Recientemente, se ha estimado que el número total de bacterias que residen en el intestino del hombre es aproximadamente igual al número de células eucariotas (del orden de 1013) (Sender et al., 2016). Las condiciones fisiológicas del TGI humano no son uniformes en todas sus posiciones y parámetros como pH, contenido de sales biliares, concentración de oxígeno o disponibilidad de nutrientes varían a lo largo de todo el sistema, lo que hace que las poblaciones microbianas varíen igualmente en número y composición en las diferentes secciones (Flint et al., 2012a; Vasapolli et al., 2019) (Figura 6). La composición de la microbiota intestinal varía también a lo largo de la vida del individuo atendiendo a factores tales como la dieta, la edad, ejercicio físico, etc., o a factores externos como el uso de antibióticos (Dominguez-Bello et al., 2010).

13 Introducción

ufc/ml

2 3 Esófago (pH<4) 10 -10 Estómago (pH~2) Bacteroides Gemella Streptococcus Megasphaera Lactobacillus Pseudomonas Prevotella Prevotella Enterococcus Rothia Helycobacter pylori Streptococcus 4-5 Veillonella < 10 Colon (pH 5-5,7) Bacteroides Intestino Delgado (pH 5-7) Clostridium Bacteroides Prevotella Porphyromonas 3 7 Clostridium 10 -10 Streptococcus Eubacterium Lactobacillus Ruminococcus ɣ-Proteobacteria Streptococcus Enterococcus Enterobacterium Enterococcus 9 12 Lactobacillus 10 -10 Peptostreptococcus Fusobacteria

Figura 6.- Esquema de la distribución de la composición microbiana a lo largo del tracto gastrointestinal humano en el que se representan los géneros más abundantes, el pH de la sección y la concentración bacteriana aproximada por gramos de contenido fecal. Adaptado de Jandhyala et al. (2015).

La microbiota intestinal del adulto es una comunidad microbiana compleja en la que se estima que más del 90% de los integrantes son bacterias; estas pertenecen por orden de abundancia a los filos Firmicutes, Bacteroidetes, Actinobacteria y Proteobacteria (Kim et al., 2017). Como poblaciones subdominantes destacan diversos grupos de los filos Verrucomicrobia y Fusobacteria (Eckburg et al., 2005). El filo Firmicutes es el grupo más abundante y diverso del TGI, y sus dos clases más representativas en este ecosistema son la clase Clostridia y la clase Bacilli. Debido a la heterogeneidad de sus miembros, la clase Clostridia se divide en 19 grupos siendo los mayoritarios los grupos XIVa y IV (Guo et al., 2020), cuyos miembros más destacados pertenecen a los géneros Ruminococcus, Clostridium, Faecalibacterium, Roseburia y Dorea. A la clase Bacilli pertenecen, entre otros,los géneros de bacterias ácido lácticas Lactobacillus, Enterococcus y Streptococcus (Kim et al., 2017). Por su parte, los componentes del filo Bacteroidetes en el TGI humano pertenecen en su mayoría a los géneros Bacteroides, Prevotella y Porphyromonas, y en menor medida a Alistipes y Parabacteroides (Johnson et al., 2017). El filo Actinobacteria, por su parte, está integrado por seis clases entre la que destaca la clase que, a su vez, contiene 34 órdenes entre los que se incluyen el orden Bifidobacteriales, en el que se integra el género Bifidobacterium, y la clase Coriobacteriia con los órdenes Coriobacteriales y Eggerthellales (Nouioui et al., 2018; Salam et al., 2020). Dentro de este último orden, destaca la familia Eggerthellaceae (Gupta et al., 2013) que incluye, entre otras, la mayor parte de las bacterias implicadas en la producción de equol (Clavel et al., 2014). El filo Proteobacteria, aunque menos abundante que los anteriores, es particularmente diverso y está representado por los géneros Desulfovibrio, Klebsiella,

14 Introducción

Shigella y Serratia, además de por la especie Escherichia coli (Rajilić-Stojanović y de Vos, 2014). De los filos subdominantes Verrucomicrobia y Fusobacteria cabe destacar, respectivamente, dos especies relacionadas con una buena salud intestinal Akkermansia muciniphila, capaz de crecer utilizando la mucina epitelial como fuente de carbono y Faecalibacterium prausnitzii, productor de butirato, compuesto con un papel crucial en la fisiología intestinal a partir de la fermentación de la fibra (Rajilić-Stojanović y de Vos, 2014). En función de los perfiles microbianos mayoritarios, los primeros estudios a gran escala utilizando técnicas de secuenciación masiva separaron las microbiotas del TGI humano en tres grupos denominados enterotipos (Arumugam et al., 2011). El enterotipo 1 se caracteriza por una abundancia de los géneros Bacteroides y Parabacteroides; en el enterotipo 2 dominan los géneros Prevotella y Desulfovibrio; y el enterotipo 3 está representado principalmente por los géneros Ruminococcus y Akkermansia (Arumugam et al., 2011). En trabajos más recientes, se han cuestionado estas agrupaciones, pues se consideran una simplificación excesiva de la diversidad microbiana de un ecosistema tan complejo y dinámico como el TGI (Knights et al., 2014). Factores como la dieta, los medicamentos, la edad o el estado de salud ejercen una fuerte influencia sobre la microbiota y, por tanto, son responsables de la variabilidad y diversidad microbianas. No obstante, la separación de la microbiota en grupos discretos de enterotipos pudiera ser relevante en el entorno clínico, por su asociación con ciertas enfermedades o para el seguimiento de intervenciones dietéticas personalizadas (Costea et al., 2018; Haddad et al., 2020). A nivel de especie, en el TGI existe una amplia diversidad microbiana, estimándose que la población humana alberga unas 1.000-1.200 especies distintas y que cada individuo posee unas 160 especies bacterianas en su intestino (Qin et al., 2010; Sankar et al., 2015). A pesar de la gran variabilidad interindividual, diversos trabajos han definido un núcleo bacteriano común de la microbiota, denominado “core”, constituido por unas 60 especies presentes en más del 50% de los individuos (Tap et al., 2009). El análisis de un amplio número de muestras fecales ha revelado que el “core” está constituido fundamentalmente por miembros de los géneros Faecalibacterium, Ruminococcus, Eubacterium, Dorea, Bacteroides, Alistipes y Bifidobacterium (Martínez et al., 2013).

2.2. Funciones de la microbiota

A lo largo de la evolución humana, la microbiota intestinal ha desarrollado una relación mutualista con el hospedador alcanzando un estado de equilibrio en el que tanto

15 Introducción hospedador como microorganismos resultan beneficiados (Hooper y Gordon, 2001). Cualquier desviación en las proporciones relativas de los microorganismos en este estado de equilibrio conduce a lo que se conoce con el término de “disbiosis”, la cual conlleva importantes consecuencias adversas para la salud (Martínez et al., 2017). La contribución de la microbiota a la homeostasis intestinal está mediada por sus actividades, entre las que se incluyen funciones metabólicas, inmunológicas y protectoras (Gentile y Weir, 2018).

2.2.1. Función metabólica

La microbiota intestinal desempeña un papel importante en el metabolismo de diversos compuestos con los que contribuye al abasteciendo de energía al hospedador, incluyendo carbohidratos, proteínas, compuestos no nutritivos y diversos micronutrientes (Gill et al., 2006). Parte de los nutrientes ingeridos con la dieta no son metabolizados por nuestras propias enzimas y alcanzan el intestino grueso intactos. Así, la fermentación de la fibra (carbohidratos no digeribles de la dieta) se lleva a cabo por bacterias de los géneros Bacteroides, Roseburia, Bifidobacterium, Faecalibacterium y otros de la familia Enterobacteriaceae (Jandhyala et al., 2015). Como resultado de esta degradación se producen ácidos grasos de cadena corta (AGCC) como butirato, propionato y acetato que actúan como fuentes de energía para los colonocitos; y otros compuestos como lactato, etanol, CO2, y H2, que son utilizados por el propio hospedador, por microorganismos fermentadores secundarios o que son excretados (Selber-Hnatiw et al., 2017). Junto con las proteasas humanas, la microbiota intestinal también está implicada en el metabolismo de las proteínas, contribuyendo a su degradación hasta aminoácidos (Jandhyala et al., 2015). Además, a través de la dieta se consumen compuestos no nutritivos, como los polifenoles, que son metabolizados en su mayor parte por la microbiota intestinal, incrementado su biodisponibilidad y actividad biológica. De la misma forma que con los componentes de la dieta, la microbiota participa también en el aprovechamiento de los constituyentes del TGI. Así, la mucina, proteína fuertemente glicosilada, se degrada por la acción de microorganismos del filo Verrucomicrobia, y en especial, como se ha dicho, por la especie A. muciniphila (Flint et al., 2012b). Por otra parte, los microorganismos de la microbiota sintetizan anaeróbicamente algunas vitaminas, como la vitamina K2 (menaquinona), que contribuye a disminuir el riesgo de enfermedades cardiovasculares y el mantenimiento de la densidad ósea, y vitaminas del grupo B, como la B5 (ácido pantoténico), B7 (biotina), B9 (ácido fólico) y B12 (cianocobalamina), que actúan como coenzimas en diversos procesos del metabolismo celular (Kho y Lal, 2018). La microbiota intestinal tiene un papel destacado

16 Introducción en el co-metabolismo, junto al hospedador, de los ácidos biliares e interviene en el metabolismo de compuestos xenobióticos (sustancias químicas sintéticas exógenas que llegan al organismos por diversas vías) como contaminantes o fármacos (Pascale et al., 2018), lo que puede tener importantes implicaciones en el tratamiento de algunas enfermedades.

2.2.2. Función inmunológica

La microbiota intestinal posee un papel destacado en el desarrollo y maduración del sistema inmune del hospedador a través de la interacción constante con las células que componen la mucosa del TGI. La microbiota contribuye tanto al desarrollo de la inmunidad innata como de la inmunidad específica. Así, participa en la maduración del tejido linfoide asociado a las mucosas, en la relación entre células T reguladoras y efectoras, en los niveles de inmunoglobulina A producida por células B, en las células linfoides innatas tipo 3, además de en el número de macrófagos residentes y células dendríticas de la lámina propia (Jandhyala et al., 2015; Jiao et al., 2020). Esto se ha puesto en evidencia en experimentos llevados a cabo con animales axénicos (carentes de microbiota intestinal) o gnotobióticos (inoculados con un solo microorganismo), los cuales muestran un sistema inmune poco desarrollado (Al-Asmakh y Zadjali, 2015).

2.2.3. Función protectora

El efecto protector directo que resulta de la interacción de la microbiota comensal con el hospedador se denomina “resistencia a la colonización” (Leshem et al., 2020). Así, existen comunidades microbianas autóctonas del intestino que ocupan un determinado nicho e impiden la colonización y el desarrollo de microorganismos patógenos como Clostridioides difficile o Helicobacter pylori (Kho y Lal, 2018). Además, la microbiota intestinal es capaz de dirigir la maquinaria de defensa del hospedador para eliminar al patógeno. De esta forma, determinados componentes celulares y metabolitos microbianos son capaces de inducir la síntesis de péptidos antimicrobianos por el hospedador, como lectinas, catelicidinas o prodefensinas (Jandhyala et al., 2015). La intercomunicación entre microbios y hospedador resulta en la activación de vías de señalización esenciales para promover la función de barrera de la mucosa y la producción de mucina e inmunoglobulina A (IgA) (Hasan y Yang, 2019). Asimismo, la producción de ácido láctico, AGCC, peróxido de hidrógeno, ácidos biliares y otras sustancias (bacteriocinas) también se ha relacionado con mecanismos de protección antimicrobiana (Leshem et al., 2020). Finalmente, la microbiota intestinal juega también un papel protector importante

17 Introducción ayudando a mantener la integridad estructural de la barrera intestinal mediante el mantenimiento de las uniones intercelulares estrechas y la reparación epitelial tras una lesión (Sekirov et al., 2010).

2.3. Factores que influyen en la microbiota

2.3.1. Microbiota y dieta

La alimentación es uno de los factores que ejercen una mayor influencia sobre la composición de la microbiota intestinal (Flint et al., 2012a). La dieta parece tener un papel fundamental para el correcto equilibrio de los microorganismos que constituyen la microbiota y, por tanto, para la buena salud del hospedador (Graf et al., 2015). El efecto de la dieta sobre la microbiota comienza tras el nacimiento: la alimentación con leche materna o de fórmula influye significativamente en la implantación, desarrollo y diversidad de la microbiota. Así, especies de los géneros Lactobacillus y Bifidobacterium son dominantes en el intestino del neonato cuando la alimentación se hace exclusivamente con leche materna, mientras que cuando se utiliza leche de fórmula los microorganismos mayoritarios pertenecen a la familia Enterobacteriaceae y a los géneros Enterococcus, Bacteroides, Clostridium y Streptococcus (Hasan y Yang, 2019). Tras la introducción de alimentos sólidos, la microbiota continúa evolucionando adquiriendo la composición, diversidad y funcionalidad habitual en el adulto. Determinados patrones de alimentación a largo plazo se han asociado con los distintos enterotipos microbianos (Wu et al 2011). Así, la dieta típica de Europa con alto contenido en proteína animal y grasa saturada se relacionaría con el enterotipo 1 (Bacteroides-Parabacteroides), mientras que una dieta rica en carbohidratos y azúcares simples parece asociarse con el enterotipo 2 (Prevotella- Desulfovibrio) característico de África (Wu et al., 2011). Dado que la dieta puede modificarse, existe un gran potencial terapéutico para manipular la diversidad, composición y estabilidad de la microbiota, influyendo también en su funcionalidad (Gentile y Weir, 2018). La mayoría de estudios en este sentido se centran en los efectos que los macronutrientes de la dieta, como proteínas, carbohidratos o lípidos, tienen sobre la modulación de la microbiota intestinal. Sin embargo, también los compuestos no nutritivos, como los polifenoles, y en concreto las isoflavonas, pueden ejercer una gran influencia sobre determinadas comunidades bacterianas (Nakatsu et al., 2014; Guadamuro et al., 2015). Con todo, los cambios microbianos pueden ser tan solo puntuales o transitorios y, por el momento, más allá de las correlaciones estadísticas, no existe una evidencia clara de que la dieta induzca cambios permanentes en la microbiota de un individuo (Leeming et al., 2019).

18 Introducción

2.3.2. Alimentación funcional, probióticos y prebióticos

El concepto de “alimentación funcional” surgió en Japón en los años 80 en un intento del gobierno de reducir los costes sanitarios asociados al envejecimiento de la población. Si bien no existe una definición universal aceptada para el término “alimentos funcionales” (AF), en general estos hacen referencia a “aquellos alimentos (bebidas incluidas) ingeridas como parte de la dieta que, más allá de sus propiedades nutricionales, contienen compuestos biológicamente activos con efectos positivos en la promoción de la salud y la prevención de enfermedades” (Diplock et al., 1998; Roberfroid, 2000). Los AF incluyen alimentos tradicionales, como el aceite de oliva o el tomate, o productos manufacturados a los que se les ha añadido algún compuesto bioactivo, como las leches enriquecidas. El término AF se utiliza en ocasiones como sinónimo de nutracéutico, aunque este término se suele reservar para componentes alimenticios administrados de forma ocasional en forma de cápsulas, comprimidos o polvos para disolver, como los suplementos de vitaminas o los comprimidos de isoflavonas soja. En la actualidad, los AF con mayor interés y más ampliamente estudiados son los que incluyen en su composición probióticos y/o prebióticos. Los probióticos se definen como “microorganismos vivos que, cuando son administrados en cantidades adecuadas, confieren un beneficio sobre la salud del hospedador” (Hill et al., 2014). La mayoría de los microorganismos probióticos pertenecen a los géneros Lactobacillus y Bifidobacterium, aunque también se han descrito efectos probióticos en cepas de otras especies bacterianas o fúngicas. Diversos estudios recientes muestran resultados prometedores con cepas de los géneros Roseburia, Akkermansia, Propionibacterium y Faecalibacterium, a los que se conoce como “probióticos de nueva generación” (Martín et al., 2018). Los mecanismos a través de los cuales los probióticos ejercen sus efectos beneficiosos sobre la salud se basan en su interacción con el hospedador y con los componentes de la microbiota. Entre los mecanismos de acción de los probióticos se encuentran: (a) la modulación del sistema inmune, (b) la interacción con la microbiota intestinal (p. ej., producción de sustancias antimicrobianas), (c) la producción de ácidos orgánicos, (d) la competencia por la adhesión al epitelio intestinal y la obtención de nutrientes, (e) la mejora de la función de barrera intestinal, (f) la interacción probiótico-hospedador mediante estructuras de la superficie celular (p. ej,. exopolisacáridos o EPS), (g) la síntesis de moléculas con efectos sistémicos (pj., ácido γ-aminobutírico o GABA) y (h) la producción de enzimas (Sanders et al., 2019). El uso de probióticos está indicado en el tratamiento de problemas intestinales con componente inflamatorio como la colitis ulcerosa, la enfermedad de Crohn o las ileítis no específicas; también en trastornos como en el síndrome del colon irritable, en el tratamiento de las diarreas (diarrea del viajero, diarrea asociada al tratamiento con

19 Introducción antibióticos) o la intolerancia a la lactosa. Los probióticos se ensayan también para la prevención del cáncer colorrectal y úlceras pépticas. En afecciones extraintestinales, los probióticos están indicados para la prevención y tratamiento de infecciones del tracto urinario (vaginosis y vaginitis), en la prevención de la obesidad, diabetes, ECVs, así como en el tratamiento de enfermedades autoinmunes (dermatitis, alergias, asma, etc.) (Markowiak y Ślizewska, 2017). El término prebiótico ha sido definido recientemente como “un sustrato que es utilizado selectivamente por los microorganismos del hospedador confiriendo un beneficio para la salud” (Gibson et al., 2017). Los carbohidratos no digeribles entre los que se incluyen la inulina, los fructooligosacaridos (FOS), los galactooligosacáridos (GOS), arabinooligosacáridos (AOS) y la lactulosa, entre otros, han sido los prebióticos más estudiados hasta la fecha. Estos compuestos son fermentados específicamente por bacterias beneficiosas presentes en el TGI favoreciendo su crecimiento y la producción de metabolitos que promueven la salud del hospedador como los AGCC acetato y butirato. Importantes funciones prebióticas se describen también para los polifenoles y otros compuestos fitoquímicos de las plantas, aunque sus efectos en la salud, por el momento, no están sustentados científicamente.

2.4. Métodos de estudio de la microbiota intestinal

En las dos últimas décadas ha habido un avance considerable en los métodos de estudio de la diversidad microbiana de los ecosistemas acuáticos y terrestres, incluyendo el del TGI humano. La microbiota intestinal se estudió en el pasado mediante estrategias de cultivo, y la identificación de microorganismos se llevaba a cabo con el estudio de su morfología y sus propiedades bioquímicas y metabólicas. El desarrollo de herramientas moleculares basadas en la reacción en cadena de la polimerasa (PCR) permitió después realizar una identificación rápida e inequívoca de los microorganismos mediante la amplificación, secuenciación y análisis del gen que codifica el ARNr 16S. La anaerobiosis estricta y los altos requerimientos nutricionales de la mayoría de los microorganismos que colonizan el TGI dificultan su cultivo in vitro (Thursby y Juge, 2017). El desarrollo y aplicación hace unas décadas de las “técnicas independientes de cultivo”, tales como la electroforesis en geles de gradiente desnaturalizantes (DGGE), la hibridación fluorescente in situ (FISH), la PCR cuantitativa en tiempo real (qPCR), etc., revelaron que en algunos ecosistemas más del 90% de los microorganismos no se desarrollaban en cultivo utilizando técnicas convencionales (Amann y Kühl, 1998). La aplicación de estas técnicas independientes de cultivo, en trabajos como el de Zoetendal et al. (2006) o Rajilić‐

20 Introducción

Stojanović et al. (2007), permitió la identificación en el TGI humano de más de 1.000 especies bacterianas diferentes. Tras el ingente esfuerzo de secuenciación que supuso el desciframiento del genoma humano, en el año 2004 aparecieron lo que se ha dado en conocer como las nuevas tecnologías de secuenciación masiva (NGS) (Fraher et al., 2012). La aplicación de las técnicas NGS ha revolucionado la microbiología, ya que permite la secuenciación de genomas bacterianos completos (genómica), la caracterización de la diversidad microbiana de un hábitat utilizando genes diana con información evolutiva (metagenómica dirigida o filogenética), o la obtención de información del conjunto de genomas bacterianos de una muestra (metagenómica no dirigida o “shotgun”) (Costa y Weese, 2019). El mejor ejemplo de ello es el proyecto del microbioma humano que permitió la caracterización de la microbiota humana de cinco nichos del cuerpo entre los que se encuentra el TGI (Peterson et al., 2009). Tras el desarrollo e implantación de la metagenómica, se han desarrollado otras técnicas “ómicas” que, de forma global, proporcionan información sobre la funcionalidad y actividad de la microbiota. Así, la metatranscriptómica, la metaproteómica y metabolómica proporcionan información, respectivamente, sobre los transcritos de ARN que se producen, las proteínas y los metabolitos generados por los microorganismos en un espacio y tiempo determinados. La gran profundidad de análisis de estas técnicas ha permitido adquirir un mayor conocimiento de la composición, diversidad y funcionalidad de la microbiota del TGI.

2.4.1. Modelos intestinales

Por razones éticas, la evaluación in vivo de los factores que afectan a la microbiota intestinal resulta muchas veces imposible, por lo que es necesario la búsqueda de métodos alternativos como el empleo de modelos intestinales in vitro y ex vivo. Los modelos intestinales in vitro tratan de imitar las condiciones fisiológicas del TGI con el fin de evaluar el efecto de los compuestos bioactivos de alimentos, fármacos, u otros sobre las poblaciones de la microbiota (Verhoeckx et al., 2015). Existen modelos intestinales capaces de simular la digestión gastrointestinal, incluyendo métodos de digestión estáticos y dinámicos. Los modelos estáticos son más simples y se utilizan fundamentalmente para estudiar el efecto sobre la microbiota de alimentos sencillos o componentes de la dieta purificados (Minekus et al. 2014). Puesto que esta metodología no es capaz de reproducir el proceso digestivo, se han desarrollado diversos modelos dinámicos del TGI que mimetizan con mayor fidelidad las condiciones fisiológicas de este (Verhoeckx et al, 2015). Estos modelos son una herramienta para el estudio de la

21 Introducción modulación de las poblaciones microbianas por la dieta y constituyen una alternativa a los ensayos con animales y a los ensayos clínicos en humanos. Como ejemplo de modelo intestinal dinámico cabe destacar el TIM-2 desarrollado en el TNO (Organización holandesa para la investigación científica aplicada). Este modelo es capaz de simular las condiciones del colon proximal humano (Minekus et al., 1999) (Figura 7). De forma concisa, este sistema está formado por cuatro unidades que actúan como simuladores independientes, rodeadas por unas paredes flexibles que recrean los movimientos peristálticos y evitan la formación de fases en el contenido luminal. El equipo, completamente computarizado, está equipado con sensores y bombas que mantienen constantes las condiciones de pH, temperatura y volumen. Un sistema de diálisis se encarga de mantener los electrolitos en suspensión y los AGCC en concentraciones fisiológicas y, además, mantiene el circuito en condiciones de anaerobiosis estricta mediante el insuflado continuo de nitrógeno.

Figura 7.- Esquema del modelo intestinal in vitro TIM-2. El sistema está integrado por: (a) compartimentos con movimientos peristálticos que albergan la materia fecal y el medio de cultivo, (b) sensor de pH, (c) bombas de álcali, (d) sistema de diálisis con membrana semipermeable, (e) sensor de nivel, (f) entrada de gas N2, (g) zona de muestreo, (h) salida de gas N2, (i) jeringuilla de alimentación y (j) sensor de temperatura. Imagen tomada de Rehman et al. (2012).

El TIM-2 ha sido ampliamente empleado como modelo intestinal en el estudio de la fermentación de carbohidratos no digeribles y su transformación en AAGC, en la evaluación del efecto de los probióticos en el restablecimiento de la microbiota tras el tratamiento con antibióticos, o bien para determinar el efecto de componentes de la dieta, como los polifenoles o las saponinas, sobre la composición y actividad metabólica de la

22 Introducción microbiota. Más recientemente, se ha utilizado también para evaluar el efecto en la microbiota de esteroles vegetales (Cuevas-Tena et al., 2019). Por su parte, los modelos intestinales ex vivo precisan de tejidos funcionales viables u órganos aislados incubados bajo condiciones controladas. Proceden fundamentalmente de animales debido a la limitada disponibilidad de tejidos humanos (Roeselers et al., 2013). El empleo de este tipo de sistemas añade complejidad a los ensayos pues considera la comunicación celular y el posible intercambio de sustancias entre distintos tipos de células, constituyendo, por tanto, un paso previo a los modelos animales, poco factibles en la mayoría de los casos.

3. MICROBIOTA Y EQUOL

A través de su maquinaría enzimática, la microbiota intestinal es responsable en parte del metabolismo de las isoflavonas de la soja. Sin embargo, no todos los individuos producen los mismos compuestos finales (p.ej., O-DMA o S-equol), de donde se deduce que la composición y actividad de la microbiota es clave en el metabolismo de las isoflavonas. Esta diversidad en la producción de metabolitos puede estar relacionada también con los beneficios de su consumo. De hecho, algunos autores especulan que sólo los individuos que poseen en su microbiota microorganismos productores de equol a partir de la daidzeína son capaces de obtener el máximo beneficio de la ingesta de isoflavonas.

3.1. Fenotipo productor de equol

En todas las especies animales estudiadas hasta el momento (gallinas, cabras, vacas, ovejas, rata, ratones, monos, chimpancés, perros y cerdos) se ha detectado equol en orina en respuesta al consumo de isoflavonas (Setchell y Clerici, 2010a; Schwen et al., 2012). Sin embargo, sólo entre el 25-50% de los humanos poseen un fenotipo productor, porcentaje que varía en función de las poblaciones y de sus hábitos alimenticios (Setchell and Cole, 2006; Bolca et al., 2007; Hall et al., 2007; Peeters et al., 2007). Un individuo se identifica como productor de equol cuando se detectan concentraciones de equol >83 nmol/L en plasma o >1000 nmol/L en orina (Setchell et al., 2002). Algunos estudios sugieren que la produción de equol es una condición bastante estable, de tal forma que los individuos mantienen su condición de productores a lo largo del tiempo (Lampe et al., 2001; Setchell et al., 2003b; Wiseman et al., 2004). En este caso, la conversión de daidzeína en equol podría estar regulada bajo algun tipo de control génetico (Atkinson et al., 2005), lo que posibilitaría que la actividad metabólica permaneciese

23 Introducción estable indefinidamente. Sin embargo, existen otros trabajos en los que se muestra una pequeña tasa de conversión de individuos productores a no productores de equol y viceversa (Frankenfeld et al., 2004), lo que sugiere que, además del control genético, los factores ambientales podrían afectar también a la estabilidad del fenotipo. Así, en un estudio en el que se evaluó el grado de incidencia del fenotipo productor y no productor en personas sanas y pacientes con cáncer de próstata se observó un porcentaje de estabilidad del fenotipo productor del 85% (Akaza et al., 2004). También se han descrito cambios en el fenotipo productor en un grupo de mujeres menopáusicas tratadas con suplementos de isoflavonas (Franke et al., 2012). A pesar de estas evidencias, los factores implicados en estos cambios en la producción de equol no se conocen por el momento.

3.1.1. Influencia de la dieta en el fenotipo productor de equol

Algunos estudios epidemiológicos sugieren que el consumo frecuente de isoflavonas y compuestos vegetales podría promover el carácter productor de equol. Así, mientras que un 20-35% de los individuos occidentales producen equol, en poblaciones orientales o en individuos que llevan una dieta vegana este porcentaje se sitúa en un 50-55% (Setchell y Cole, 2006). En los individuos productores, otros componentes de la dieta pudieran influir directa o indirectamente en la magnitud de la producción de equol. En este sentido, la producción de equol se asocia con la presencia en heces de propionato y butirato, lo que sugiere que una dieta rica en carbohidratos podría estimular la produción (Decroos et al., 2005; Cassidy et al., 2006). Sin embargo, los resultados sobre ingesta de FOS y la producción de equol son contradictorios. Algunos autores señalan que su presencia inhibe la producción de equol (Decroos et al., 2005), mientras que otros determinan un incremento de la producción (Lipovac et al., 2015). El consumo combinado de daidzeína junto con almidón resistente (Tousen et al., 2011a) o con lactulosa (Zheng et al., 2014) también se ha asociado con un incremento de producción de equol. Igualmente, el consumo de leche y productos lácteos junto con daidzeína parece correlacionarse positivamente con la cantidad de equol excretado en orina (Frankenfeld, 2011a). Todos estos trabajos ponen de manifiesto la importancia de los hábitos alimenticios en relación con la produción de equol, así como su posible asociación con la estabilidad del fenotipo productor. Además de con la dieta, el uso de determinados antibióticos se ha relacionado también con una mayor o menor producción de equol, lo que sugiere de nuevo la existencia de diferencias interindividuales en los biotipos intestinales productores (Atkinson et al., 2004).

24 Introducción

3.2. Microorganismos productores de equol

La daidzina se desglicosila dando lugar a la daidzeína y esta, a su vez, por acción de determinadas bacterias intestinales se transforma en S-(-)equol en los individuos productores (Tabla 1). El conocimiento que se tiene sobre los microorganismos capaces de llevar a cabo esta transformación, sin embargo, es aún limitado (Clavel y Mapesa, 2013). Algunos microorganismos son capaces de metabolizar las isoflavonas hasta alguno de los compuestos intermediarios o O-DMA, pero no son capaces de producir S-equol (Tabla 1).

Tabla 1.- Bacterias involucradas en el metabolismo de daidzeína y del intermediario dihidrodaidzeína (DHD) y la consecuente producción de los metabolitos derivados finales S-equol y O-desmetilangolensina (O-DMA).

Especie Cepa Origen Referencia

Daidzeína  DHD  S-equol Adlercreutzia equolifaciens FJC-B9T (=JCM 14793T=DSM Heces humanas Maruo et al. (2008) 19450T=CCUG 54925T) FJC-B12 FJC-B15 FJC-B19 FJC-B20 FJC-D47 FJC-D53 Asaccharobacter celatus do03T(=JCM 14811T=DSM Ciego de rata Minamida et al. (2006) 18785T=AHU 1763T) Bifidobacterium breve ATCC 15700T Heces humanas Elghali et al. (2012) Bifidobacterium longum BB536 Heces humanas Elghali et al. (2012) Catenibacterium sp. D2 Heces de cerdo Yu et al. (2008) Eggerthella sp. YY7918 Heces humanas Yokoyama y Suzuki(2008) Eggerthella sp. D1 Heces de cerdo Yu et al. (2008) Eggerthella-like sp. SNR48-44 Tofu Abiru et al. (2013) SNR44-10 SNR46-41 SNR45-571 SNR48-350 Enterorhabdus mucosicola Mt1B8T,a Mucosa ileal de ratón Matthies et al. (2008) Lactobacillus casei/paracasei CS2 (JS1) Heces humanas Kwon et al. (2018) Lactobacillus sakei/graminis CS3 Heces humanas Kwon et al. (2018) Lactobacillus intestinalis JCM 7548 Heces de rata Heng et al. (2019) Lactococcus garvieae 20-92 Heces humanas Uchiyama et al. (2007) Paraeggerthella sp. SNR40-432 Tofu Abiru et al. (2013) Pediococcus pentosaceus CS1 Heces humanas Kwon et al. (2018) Proteus mirabilis LH-52 Intestino de rata Guo et al. (2012) Slackia equolifaciens DZET (=JCM 16059T= CCUG Heces humanas Jin et al. (2010) 58231T) Slackia isoflavoniconvertens HE8T,a (=DSM 22006T,a) Heces humanas Matthies et al. (2009) Slackia sp. NATTS Heces humanas Tsuji et al. (2010)

Daidzeina  DHD  O-DMA Clostridium sp. HGH 136 Heces humanas Hur et al. (2002) Clostridium sp. SY8519 Heces humanas Yokoyama et al. (2010) Clostridium sp. Aeroto-AUH-JLC108 Heces de gallo Li et al. (2015) (=CGMCC 9550) Enterococcus faecium INIA P553 - Gaya et al. (2018) Enterococcus hirae AUH-HM195 Heces de faisán Yu et al. (2009) Eubacterium ramulus wK1 Heces humanas Schoefer et al. (2002) E. ramulus Julong 601 Heces humanas Wang et al. (2004)

25 Introducción

Tabla 1.-Continuación

Especie Cepa Origen Referencia

Daidzeína  DHD Clostridium sp. HGH6 Heces humanas Hur et al. (2000) Clostridium-like TM-40 Heces humanas Tamura et al. (2007) Lactobacillus sp. Niu-O16 Rumen bovino Wang et al. (2005a)

DHD  S-equol A. equolifaciens FJC-A10 Heces humanas Maruo et al. (2008) FJC-A161 Eggerthella sp. Julong 732 Heces humanas Wang et al. (2005b) “-“: origen desconocido. a Estas cepas producen además 5-hidroxi-equol a partir de la genisteína. El superíndice T señala las cepas que son cepas tipo de la especie.

Así, las cepas bacterianas Clostridium TM-40 y HGH6 son capaces de metabolizar la daidzeína generando DHD (Hur et al., 2000; Tamura et al., 2007). También se ha descrito la producción de equol mediante la combinación de dos cepas bacterianas: Lactobacillus sp. Niu-O16, que convierte la daidzeína en DHD, y Eggerthella sp. Julong 732, que metaboliza la DHD hasta equol (Wang et al., 2007). Incluso, se han caracterizado consorcios en los que diferentes microorganismos contribuyen a la formación de equol mediante reacciones enzimáticas complementarias Así, Decross et al. (2005) describieron una comunidad productora de equol estable integrada por cuatro especies bacterianas: Lactobacillus mucosae EPI2, Enterococcus faecium EPI1, Finegoldia magna EPI3 y Veillonella sp. EP. En las últimas décadas, se han identificado diversas cepas intestinales de procedencia animal y humana capaces de transformar la daidzeína en equol. La mayoría de los aislados productores descritos hasta el momento pertenecen a la clase Coriobacteriia del filo Actinobacteria (Salam et al., 2020). La clase Coriobacteriia está integrada por bacterias Gram-positivas, anaerobias estrictas y con grandes requerimientos nutricionales. Incluye dos órdenes Coriobacteriales y Eggerthellales (Gupta et al., 2013), con las familias Coriobacteriaceae y Atopobiaceae en el primer orden y la familia Eggerthellaceae en el segundo (Gupta et al., 2013; Nouioui et al., 2018) (Figura 8). La mayor parte de los microorganismos productores de equol de origen intestinal han quedado englobados en esta última familia. La familia Eggerthellaceae comprende los géneros Eggerthella, Adlercreutzia, Asaccharobacter, Cryptobacterium, Denitrobacterium, Ellagibacter, Enterorhabdus, Enteroscipio, Gordonibacter, Paraeggerthella, Parvibacter, Rubneribacter, Senegalimassilia y Slackia (Salam et al., 2020). Dada la similitud de las cepas analizadas, los géneros Asaccharobacter, Enterorhabdus y Parvibacter podrían ser sinónimos de Adlercreutzia, el primero de los descritos (Nouioui et al., 2018). Las cepas productoras de equol de origen intestinal pertenecen a las especies Adlercreutzia equolifaciens (Maruo et al., 2008), Asaccharobacter celatus (Minamida et al., 2008), Enterorhabdus mucosicola

26 Introducción

(Matthies et al., 2008), Slackia equolifaciens (Jin et al., 2010) y Slackia isoflavoniconvertens (Matthies et al., 2009). Algunas de estas cepas, como S. isoflavoniconvertens HE8T y E. mucosicola Mt1B8T, son capaces también de producir 5-hidroxiequol a partir de la genisteína (Matthies et al., 2008, 2009). Otros aislados productores de equol han sido identificados únicamente a nivel de género, como las cepas Slackia sp. NATTS (Tsuji et al., 2010), Eggerthella sp. YY7918 (Yokoyama y Suzuki, 2008), Eggerthella sp. D1(Yu et al., 2008) o Paraeggerthella sp. SNR40-432 (Abiru et al., 2013). También, en este último trabajo se obtuvieron cinco cepas bacterianas capaces de producir equol SNR48-44 (grupo II) y SNR44-10, SNR45-571, SNR46-41 y SNR48-350 (grupo III) que mostraron una homología en el gen que codifica el ARNr 16S del 92% con las especies Eggerthella sinensis y Eggerthella lenta, respectivamente (Abiru et al., 2013), lo que sugiere que cada grupo pudiera representar un nuevo género dentro de la familia Eggerthellaceae. A pesar de todo este conocimiento, por el momento no está claro aún si la capacidad de producir equol dentro de la familia Eggerthellaceae es una característica específica de género, de especie o de cepa (Mayo et al., 2019).

Slackia piriformis YIT 12062 Slackia heliotrinireducens DSM 20476 10 T 0 10 Slackia exigua ATCC700122 0 Cryptobacterium curtum DSM 15641T 10 T 10 Denitrobacterium detoxificans DSM21843 0 T Orden 0 Senegalimassilia anaerobia JC110 Familia T 10 Eggerthellales 10 Eggerthella lenta DSM2243 Eggerthellaceae 0 0 10 Gordonibacter pamelaeae 7-10-1-b 0 T 10 Enterorhabdus mucosicola DSM19490 0 10 Enterorhabdus caecimuris B7 T 10 0 Adlercreutzia equolifaciens DSM19450 10 0 T 0 10 Assacharobacter celatus DSM18785 0 Coriobacterium glomerans DSM20642T Enorma massiliensis phIT 10 10 Enorma timonensis GD5 0 0 T 10 Collinsella aerofaciens ATCC 25986 Familia 0 Collinsella tanakaei YIT 12063 Coriobacteriaceae 10 10 Collinsella stercoris DSM 13279 0 0 10 Orden 0 10 Collinsella intestinalis DSM 13280 0 Coriobacteriales Atopobium vaginae DSM 15829 Atopobium fossor DSM 15642 10 10 Atopobium minutum DSM 20586T 0 0 Familia 99 Atopobium parvulum DSM 20469 10 Atopobium rimae ATCC 49626 Atopobiaceae 0 10 Olsenella scatoligenes SK9K4 0 98 Olsenella umbonata DSM 22620 99 Olsenella uli DSM 7084

Figura 8.- Árbol filogenético de la clase Coriobacteriia dentro del filo Actinobacteria adaptado de Nouioui et al. (2018). La primera rama incluye el órden Eggerthellales (verde claro) con la familia Eggerthellaceae (verde) y en la segunda rama el órden Coriobacteriales (naranja claro) integrado por las familias Coriobacteriaceae (amarillo) y Atopobiaceae (naranja). El superíndice “T” hace referencia a la cepa tipo. DSMZ: Colección alemana de microorganismos y cultivos celulares; ATTC: Colección americana de cultivos tipo, YIT: Instituto de tecnología de Yeoju.

Además de las bacterias pertenecientes a la familia Eggerthellaceae, se han identificado microorganismos productores de equol pertenecientes a otros grupos bacterianos. Así, producen equol a partir de daidzeína las cepas Proteus mirabilis LH-52 (Guo et al., 2012) y

27 Introducción

Catenibacterium sp. D2 (Yu et al., 2008). Además de L. garvieae 20- 92 (Uchiyama et al., 2007), de forma reciente se han identificado otras cepas de bacterias ácido lácticas (BAL) productoras de equol, como Pediococcus pentosaceus CS1 (Kwon et al., 2018), Lactobacillus paracasei CS2 (JS1), Lactobacillus sakei/Lactobacillus graminis CS3 y Lactobacillus intestinalis JCM 7548 (Kwon et al., 2018; Heng et al., 2019). Finalmente, se han encontrado también dos cepas productoras del género Bifidobacterium: Bifidobacterium breve ATCC 15700T y Bifidobacterium longum BB536 (Elghali et al., 2012).

3.3. Caracterización molecular de la formación de equol

La síntesis de equol a partir de daidzeína se lleva a cabo mediante la acción consecutiva de tres reacciones enzimáticas de reducción a través de los compuestos intermediarios DHD y THD (Schröder et al., 2013). La identificación de los enzimas implicados se llevó a cabo por primera vez en la cepa Lactococcus garvieae 20-92 (Shimada et al., 2010, 2011, 2012). En esta cepa, se detectaron tres genes esenciales para la formación de equol que codifican reductasas: el gen dzr que codifica la daidzeína reductasa dependiente de NADP(H), involucrada en la conversión enantioselectiva de daidzeína en S-dihidrodaizeína (S-DHD) (Shimada et al., 2010), el gen ddr que codifica para la dihidrodaizeína reductasa, encargada de la transformación de DHD en TDH, y el gen tdr que codifica para la tetrahidrodaidzeína reductasa, que interviene en el paso final de formación de equol a partir de la TDH (Shimada et al., 2011). Posteriormente, se identificó un cuarto gen en L. garviae 20-92, ubicado en el clúster del equol y precediendo a las reductasas que codifica una dihidrodaidzeína racemasa. Este enzima no es esencial, pero su presencia permite una producción más eficiente de equol (Shimada et al., 2012). Todos estos genes se encontraban formando parte de un operón de unas 10 kpb (Shimada et al., 2010, 2011, 2012). La aplicación de las técnicas NGS ha permitido la caracterización de los genomas de varias cepas productoras de equol, lo que resulta de gran ayuda para conocer y comparar las bases genéticas involucradas en su síntesis. En S. isoflavoniconvertens, al igual que en Slackia sp. NATTS, la secuencia aminoacídica de las proteínas codificadas por los genes dzr, ddr y tdr muestran una alta homología con las identificadas en L. garvieae (Schröder et al., 2013; Tsuji et al., 2012). Igualmente, el análisis de los genomas de A. equolifaciens DSM 19450T y de Eggerthella sp. YY918 revelaron también la presencia de genes similares codificando las tres reductasas (Toh et al., 2013; Kawada et al., 2016). La gran homología en las secuencias nucleotídicas y en la organización genética del clúster de biosíntesis de equol en L. garvieae y en las especies de la familia Eggerthellaceae sugiere la transferencia

28 Introducción horizontal de los genes a L. garvieae desde algún miembro de la familia (Schröder et al. 2013). Esta teoría se apoya en el contenido GC de los genes de producción de equol en L. garvieae que es del 68% (Kawada et al., 2016), muy superior al del resto de su genoma (39%) y próximo al contenido GC de los genomas de las especies de la familia Eggerthellaceae (60%) (Clavel et al. 2014). Los estudios realizados con E. mucosicola Mt1B8 y S. isoflavoniconvertens HE8 señalan una expresión inducible de las reductasas en presencia de isoflavonas en el medio de cultivo (Matthies et al., 2008, 2009). En S. isoflavoniconvertens, la presencia de daidzeína provoca la sobreexpresión de otros cinco genes del operón además de aquellos que codifican las reductasas (Schröder et al., 2013). Esta regulación coordinada sugiere su participación en el metabolismo de las isoflavonas y/o en la producción de equol. Con excepción de L. garvieae, el marco genético y las rutas bioquímicas implicadas en la producción de equol en cepas que no pertenecen a la familia Eggerthellaceae aún no se ha determinado.

3.4. Producción biotecnológica de equol

Las bacterias con mayor capacidad de producir equol pertenecen a la familia Eggerthellaceae cuyos miembros se caracterizan por requerir tiempos de incubación prolongados, una anaerobiosis estricta y altos requerimientos nutricionales (Clavel et al., 2014), cualidades que dificultan la producción biotecnológica del equol a escala industrial. Una estrategia para superar los problemas asociados a la presencia de oxígeno en las fermentaciones industriales, podría ser la “domesticación aeróbica” de las cepas productoras. Esta estrategia es la que han seguido Zhao et al. (2011) para convertir en aerotolerante la cepa Lactobacillus sp. Niu-O16. Esta cepa, sin embargo, no produce equol sino que convierte daidzeína y genisteína, respectivamente, en DHD y dihidrogenisteína. Otra forma de solventar estas limitaciones es mediante la utilización de técnicas de ingeniería genética que permitan la clonación y expresión de la maquinaria de los organismos productores en microorganismos modelo como E. coli. En este sentido, los genes de L. garvieae que codifican las tres reductasas esenciales se clonaron y se expresaron correctamente en esta bacteria (Shimada et al., 2010, 2011). En E. coli se expresaron también los genes equivalentes de S. isoflavoniconvertens HE8T (Schröder et al., 2013) y de Eggerthella sp. YY7918 (Kawada et al., 2016). Algunas mutaciones en el gen ddr parecen estabilizar los clones e incrementar la producción de equol en el hospedador heterólogo (Lee et al., 2016). Otras mutaciones en E. coli también confieren una mayor producción de equol (Lee et al., 2016). La producción en E. coli se ha incrementado

29 Introducción también aumentando la solubilidad de las isoflavonas mediante la adicción de polímeros hidrofílicos a los cultivos (Lee et al., 2018). La producción biotecnológica de equol a gran escala posibilitaría la realización de un mayor número de ensayos de intervención para evaluar de forma convincente los beneficios del equol en la salud.

3.5. Equol y alimentos funcionales

Debido al alto valor biológico de las proteínas de la soja, desde el año 1999 la Agencia Americana de Medicamentos y Alimentación (Food and Drug Administration, FDA) permite a los productos derivados llevar la alegación funcional de “beneficiosos para las enfermedades cardiacas” si las raciones contienen al menos 6,25 g de proteína (FDA, 1999). La suplementación de alimentos con equol podría dar lugar también a nuevos alimentos funcionales. Esta suplementación es aún más importante dado que no todos los individuos de la población, como hemos visto, son capaces de producir equol de forma endógena. Otra estrategia pudiera ser la transferencia de la maquinaria genética de producción de equol a bacterias alimentarias, de manera que los alimentos se enriquezcan en el bioactivo durante los procesos de elaboración; p. ej., durante la fermentación. En este sentido, la producción de equol por especies de BAL recombinantes capaces de desarrollarse adecuadamente en extractos de soja (Delgado et al., 2019), posibilitaría la producción de equol en los productos de soja fermentados. Dadas las grandes limitaciones legales del empleo de microorganismos recombinantes en alimentación, más que para la producción de alimentos, estas bacterias podrían servir para una producción “segura” de equol.

30

OBJETIVOS OBJECTIVES Objetivos

OBJETIVOS

Las isoflavonas y sus metabolitos se relacionan con numerosos efectos beneficiosos para la salud humana, incluyendo una menor incidencia de enfermedades dependientes de hormonas, asociadas al envejecimiento, neurodegenerativas, cardiovasculares y ciertos tipos de cáncer. Con todo, la utilidad más explorada de estos compuestos es la del tratamiento de los síntomas de la menopausia. El metabolismo de las isoflavonas tiene lugar por la acción de enzimas tisulares y enzimas de la microbiota intestinal, mediante las que las isoflavonas de la dieta se convierten en metabolitos más biodisponibles y ocasionalmente biológicamente más activos, como el equol a partir de la daidzeína. Este compuesto es el metabolito microbiano de las isoflavonas con mayor efecto estrogénico, mayor actividad antioxidante y con unas propiedades antiandrogénicas únicas. Sin embargo, sólo una parte de los humanos, entre un 25-50% de los individuos, es capaz de producir este compuesto bioactivo. Como consecuencia, es posible que sólo los individuos que albergan en su TGI los microorganismos que hacen posible la transformación de la daidzeína en equol puedan beneficiarse por completo del consumo de isoflavonas.

A pesar de las numerosas evidencias epidemiológicas de los beneficios de las isoflavonas y del equol sobre la salud, el conocimiento de su metabolismo y las poblaciones microbianas intestinales implicadas es aún escaso. La microbiota humana es un ecosistema complejo y dinámico compuesto mayoritariamente por microorganismos con grandes requerimientos nutricionales y estrictas condiciones de anaerobiosis. A pesar de ello, en las últimas décadas, se han identificado bacterias intestinales capaces de producir equol de la daidzeína; la mayoría pertenecen a la clase Coriobacteriia. Estos microorganismos forman parte de poblaciones subdominantes en el TGI, por lo que son difíciles de aislar del resto de componentes de la microbiota. Los estudios moleculares más recientes, llevados a cabo mediante diversas técnicas ómicas de última generación, constatan una gran diversidad interindividual de la microbiota, lo que sugiere la posibilidad de que existan microorganismos involucrados en el metabolismo de las isoflavonas y la producción de equol distintos a los descritos hasta ahora. El desarrollo y aplicación de métodos y técnicas para el estudio de la composición y estructura de estas poblaciones y su interacción con el resto de comunidades intestinales resulta determinante para su modulación, con el objetivo último de favorecer el desarrollo de poblaciones beneficiosas en relación a las isoflavonas y, en su caso a la producción de equol, y extender los efectos de estos compuestos en la salud humana.

31 Objetivos

La alimentación es uno de los factores que ejerce una mayor influencia sobre la composición de la microbiota intestinal. Actualmente, no se sabe con exactitud cómo influyen los componentes de la dieta sobre las poblaciones mayoritarias del TGI, y menos aún cuáles de estos factores tienen un efecto sobre las poblaciones que participan en el metabolismo de las isoflavonas y en la producción de equol. Algunos estudios epidemiológicos sugieren que el consumo frecuente de isoflavonas y otros polifenoles pudiera promover el carácter productor de equol, ya que, mientras que un 20-35% de los individuos occidentales producen equol, en poblaciones orientales o en individuos que llevan una dieta vegana este porcentaje se sitúa en 50-55%. Además, también se especula con el efecto que algunos componentes de la dieta pudieran tener en la magnitud de la producción de equol en individuos productores, como los fructooligosacáridos, el almidón resistente, los productos lácteos y otros. Determinar la vinculación que existe entre las poblaciones bacterianas de la microbiota intestinal, la dieta, el metabolismo de las isoflavonas y la producción de equol posibilitará el diseño de estrategias destinadas a incrementar la producción endógena de equol.

Además de los biotipos microbianos implicados, resulta determinante también el estudio de los enzimas y las rutas metabólicas que participan en el metabolismo de las isoflavonas y conducen a la formación de equol. La transformación de daidzeína en equol, a través de los intermediarios dihidrodaidzeína y tetrahidrodaidzeína, requiere la acción de tres reductasas: daidzeína reductasa, dihidrodaizeína reductasa y tetrahidrodaidzeína reductasa. Análisis genómicos recientes han revelado la presencia de un agrupamiento de genes (“cluster”), entre los que se encuentran los que codifican las reductasas, con una organización génica muy similar en todas las cepas productoras. A pesar de estos estudios, no se conoce con exactitud la ruta metabólica completa de la síntesis de equol, los mecanismos de su regulación, ni la función de muchos de los genes del cluster. Profundizar en este conocimiento permitirá potenciar los mecanismos de producción endógena de equol y desarrollar metodologías biotecnológicas para la producción de este bioactivo.

Con estos antecedentes, el objetivo general de esta Tesis es profundizar en el estudio de las interacciones de las isoflavonas de la soja con las poblaciones microbianas intestinales, con especial hincapié en aquellas involucradas en su metabolismo y en la producción de equol. Con ello contribuiremos a la caracterización taxonómica y funcional de la microbiota del intestino y a relacionar, en el complejo ecosistema del TGI, taxones o grupos poblacionales con su función metabólica específica sobre las isoflavonas. El conocimiento adquirido puede ser de gran ayuda para incrementar los beneficios en la salud del consumo de soja y suplementos de isoflavonas y extender estos beneficios a la

32 Objetivos población en general con independencia de los taxones presentes en su microbiota. Los objetivos generales se piensan alcanzar a través de la consecución de los objetivos específicos que se relacionan a continuación:

OBJETIVO 1.- Desarrollar métodos para identificar y cuantificar poblaciones intestinales involucradas en el metabolismo de las isoflavonas y la producción de equol.

OBJETIVO 2.- Estudiar las relaciones e interacciones entre isoflavonas, equol y poblaciones bacterianas intestinales.

OBJETIVO 3.- Caracterizar la producción de equol en muestras fecales y bacterias productoras con el fin de maximizar su formación endógena y biotecnológica.

33 Objectives

OBJECTIVES

Isoflavones and isoflavone-derived metabolites have been associated with several beneficial health effects: a lower incidence of hormone-dependent diseases and aging- associated disorders, a reduced risk of developing neurodegenerative and cardiovascular diseases and certain types of cancer. Nevertheless, isoflavones have been mainly acknowledged for alleviating menopause symptoms. Isoflavones metabolism is accomplished by the action of cellular enzymes along with enzymes from the human gut microbiota. Dietary isoflavones are transformed into more bioavailable metabolites and in some cases, biologically active compounds such as equol from daidzein. Equol is the isoflavone-derived metabolite, exclusively obtained from the microbial metabolism, with the greatest oestrogenic effects, the strongest antioxidant activity and unique anti- androgenic properties. However, this bioactive compound is produced by only 25-50% of human subjects and consequently, only those individuals who harbour equol-producing microorganisms in their intestines are the ones who fully benefit from the isoflavones intake.

Despite all the epidemiological evidences related to isoflavones and equol production benefits over human health, the knowledge about isoflavones metabolism and the actual bacterial populations involved is still scarce. Human gastrointestinal microbiota is a complex and dynamic ecosystem principally composed by microorganisms with highly nutritional requirements and strictly anaerobe conditions. However, during the last decades, a number of bacterial strains having a role in the isoflavones metabolism have been isolated and identified; most of them as members of the Coriobacteriia class. Since they belong to subdominant groups, the isolation of those microorganisms from other microbial biotypes present in the GI tract remains somehow challenging. In some recent studies, the use of high-throughput omics technologies has shown a large interindividual diversity which leads to the plausible existence of microorganisms involved in the isoflavones metabolism and capable of producing equol different from the ones described so far. The development and applicability of methods and techniques, which enables the study of the composition and structure of those intestinal populations and the interaction with members of other bacterial communities, turn to be decisive in an attempt to modulate human gut microbiota. On account of this, beneficial populations involved in isoflavones metabolism and equol production would be promoted and

34 Objectives consequently the contribution of these compounds would be extended to the human health.

Diet has been shown to be a pivotal factor over the composition of the intestinal microbiota. Little is known about how dietary constituents have influence on the dominant microbial populations present in the GI tract and even less, which factors have a stronger impact on populations involved in the isoflavones metabolism and in the equol production. Some epidemiological studies suggest that frequent isoflavones and other polyphenols intake could promote the equol-producer status. Therefore, whereas 20-35 % of individuals in Western populations have the ability to produce equol, in Asian regions or among vegetarians that percentage increases to 20-55%. Additionally, some dietary components are speculated to stimulate the equol production in equol producers such as fructooligosaccharides, resistant starch or some daily products. The relation among bacterial populations within the intestinal microbiota, diet, isoflavones metabolism and equol production would allow developing strategies to enhance equol production.

Besides the microbial biotypes, bacterial enzymes and metabolic pathways involved in the isoflavones metabolism and in the equol synthesis are also crucial. Daidzein is transformed into equol via the production of the intermediate compounds dihydrodaidzein and tetrahydrodaidzein and due to the action of three consecutive reductases namely daidzen reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase. Recent genomic analyses have revealed the presence of a cluster of genes, including the genes encoding the three above reductases, with similar genetic organization in all equol-producing strains. Despite these studies, metabolic pathways involved in the equol biosynthesis, their regulation mechanisms and the function of most genes within the equol cluster remain still under study. Getting a deeply insight into this knowledge would enhance mechanisms for the endogenous equol synthesis, and would develop or improve biotechnological strategies in order to maximize the production of this metabolite.

In this context, the main aim of this Thesis is to the study the interaction between soya isoflavones and intestinal microbial populations, especially the microorganisms involved in isoflavones metabolism and equol synthesis. With this purpose, the obtained results will contribute to the taxonomic and functional characterization of intestinal microbiota as well as to relate, in the complex intestinal ecosystem, taxons or population groups to their specific metabolic functions over isoflavones. All knowledge gathered over these studies could be valuable in order to promote health benefits associated to the consumption of soya and isoflavone supplements and ultimately to extend those benefits to the general

35 Objectives population independently of the taxon included in their microbiota. The general objectives are to be fulfilled by the achievement of the partial and specific objectives shown below:

OBJECTIVE 1.- To develop methods to identify and quantify intestinal population involved in isoflavones metabolism and equol production.

OBJECTIVE 2.- To study the relations and interaction among isoflavones, equol and intestinal bacterial populations.

OBJECTIVE 3.- To characterize equol production in faecal samples and equol-producing bacteria in order to maximize the endogenous and biotechnological equol synthesis.

36

TRABAJO EXPERIMENTAL EXPERIMENTAL WORK

CAPÍTULO 1

CAPÍTULO 1

Desarrollo de métodos para identificar y cuantificar poblaciones intestinales involucradas en el metabolismo de las isoflavonas y la producción de equol.

Dado que las poblaciones implicadas en el metabolismo de las isoflavonas son minoritarias en la microbiota del TGI, el desarrollo de metodologías independientes de cultivo que permitan su identificación y monitorización directamente en muestras complejas (heces o cultivos fecales) es un objetivo prioritario. En este sentido, en el Capítulo 1 se incluyen dos artículos que exponen los resultados de la aplicación de las técnicas de PCR cuantitativa a tiempo real (qPCR) y metagenómica no dirigida (“shotgun”).

. Artículo I.- Vázquez, L., Guadamuro, L., Giganto, F., Mayo, B. y Flórez, A. B. (2017). Development and use of a real-time quantitative PCR method for detecting and quantifying equol-producing bacteria in human faecal samples and slurry cultures. Frontiers in Microbiology, 8, 1–11. doi:10.3389/fmicb.2017.01155.

. Manuscrito II.- Composition and functionality of faecal microbiota from equol producer and equol non-producer women as assessed by shotgun metagenomics.

En el primer artículo se describe el desarrollo y validación de la técnica de qPCR para la detección y cuantificación de cepas productoras de equol en heces y cultivos fecales, mediante la amplificación de regiones conservadas de dos genes (ddr y tdr) que codifican reductasas implicadas directamente en la biosíntesis de este compuesto bioactivo. La técnica de qPCR desarrollada resultó altamente específica, sensible y fiable para la detección y cuantificación de bacterias productoras de equol. El gen tdr se detectó en todas las mujeres productoras en un número aproximado de copias de entre 104 y 105 por gramo. Mientras, que el gen ddr solo se amplificó en dos de tres muestras de heces de mujeres con fenotipo productor. De forma sorprendente, los dos genes se amplificaron también en dos muestras de mujeres no productoras. La detección simultánea del equol por cromatografía liquida de ultra-alta resolución (UHPLC) podría complementar la monitorización de los microorganismos intestinales productores de equol, al combinar aspectos genéticos y bioquímicos.

En un segundo artículo se describe la aplicación de la secuenciación metagenómica no dirigida con el mismo propósito, identificar y cuantificar las bacterias productoras de equol. Dada la profundidad de análisis de esta técnica se podrían resolver las

37 CAPÍTULO 1

contradicciones detectadas mediante la técnica de qPCR. A pesar de los bajos niveles en los que se encuentran los microorganismos productores de equol en el TGI, mediante metagenómica “total” se identificaron secuencias homólogas a Adlercreutzia equolifaciens y Eggerthella sp., especies a las que pertenecen cepas productoras de equol. En consonancia con los resultados de qPCR, taxones de estos tipos se detectaron tanto en la microbiota de las mujeres productoras como no productoras. La caracterización funcional de las secuencias de mujeres productoras de equol mostró un incremento en genes que codifican enzimas del ciclo de Krebs, glicosil-hidrolasas, u otros relacionados con la de- hidrogenación de compuestos y actividad histidín-kinasa en la microbiota de las mujeres. Sin embargo, debido posiblemente a su bajo número de copias respecto de otras pertenecientes a las poblaciones mayoritarias, tan solo se detectó un gen en una sola muestra relacionado directamente con la formación de equol.

38 CAPÍTULO 1

ORIGINAL RESEARCH published: 30 June 2017 doi: 10.3389/fmicb.2017.01155

Development and Use of a Real-Time Quantitative PCR Method for Detecting and Quantifying Equol-Producing Bacteria in Human Faecal Samples and Slurry Cultures

Lucía Vázquez 1, Lucía Guadamuro 1, Froilán Giganto 2, Baltasar Mayo 1 and Ana B. Flórez 1*

1 Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias, Consejo Superior de Investigaciones Científicas, IPLA-CSIC, Villaviciosa, Spain, 2 Servicio Digestivo, Hospital Universitario Central de Asturias, Oviedo, Spain

This work introduces a novel real-time quantitative PCR (qPCR) protocol for detecting and quantifying equol-producing bacteria. To this end, two sets of primers targeting the dihydrodaidzein reductase (ddr) and tetrahydrodaidzein reductase (tdr) genes, which are involved in the synthesis of equol, were designed. The primers showed Edited by: David Rodriguez-Lazaro, high specificity and sensitivity when used to examine DNA from control bacteria, University of Burgos, Spain such as Slackia isoflavoniconvertens, Slackia equolifaciens, Asaccharobacter celatus, Reviewed by: Adlercreutzia equolifaciens, and Enterorhabdus mucosicola. To demonstrate the validity Ricardo Santos, and reliability of the protocol, it was used to detect and quantify equol-producing Instituto Superior Tecnico, Portugal Anna Carratalà, bacteria in human faecal samples and their derived slurry cultures. These samples were École Polytechnique Fédérale de provided by 18 menopausal women under treatment of menopause symptoms with Lausanne, Switzerland a soy isoflavone concentrate, among whom three were known to be equol-producers *Correspondence: Ana B. Flórez given the prior detection of the molecule in their urine. The tdr gene was detected in abfl[email protected] the faeces of all these equol-producing women at about 4–5 log10 copies per gram of faeces. In contrast, the ddr gene was only amplified in the faecal samples of two of these Specialty section: This article was submitted to three women, suggesting the presence in the non-amplified sample of reductase genes Food Microbiology, unrelated to those known to be involved in equol formation and used for primer design a section of the journal in this study. When tdr and ddr were present in the same sample, similar copy numbers Frontiers in Microbiology of the two genes were recorded. However, no significant increase in the copy number Received: 10 April 2017 Accepted: 07 June 2017 of equol-related genes along isoflavone treatment was observed. Surprisingly, positive Published: 30 June 2017 amplification for both tdr and ddr genes was obtained in faecal samples and derived Citation: slurry cultures from two non-equol producing women, suggesting the genes could be Vázquez L, Guadamuro L, Giganto F, non-functional or the daidzein metabolized to other compounds in samples from these Mayo B and Flórez AB (2017) Development and Use of a Real-Time two women. This novel qPCR tool provides a technique for monitoring gut microbes that Quantitative PCR Method for produce equol in . Monitoring equol-producing bacteria in the human gut could Detecting and Quantifying Equol-Producing Bacteria in Human provide a means of evaluating strategies aimed at increasing the endogenous formation Faecal Samples and Slurry Cultures. of this bioactive compound. Front. Microbiol. 8:1155. doi: 10.3389/fmicb.2017.01155 Keywords: real time quantitative PCR, qPCR, soy isoflavones, equol, intestinal microbiology, faecal microbiota

Frontiers in Microbiology | www.frontiersin.org June 2017 | Volume 8 | Article 1155 39 CAPÍTULO 1 Vázquez et al. qPCR Quantification of Equol-Producing Bacteria

INTRODUCTION difficult to isolate from other intestinal microbes. Some qPCR methods for detecting and quantifying Coriobacteriaceae species Epidemiological evidence suggests high intakes of soy foods in faecal samples have already been developed (Harmsen et al., or purified soy isoflavones to be associated with less intense 2000; Thorasin et al., 2015; Cho et al., 2016). However, as the menopause symptoms and a reduced risk of developing amplification primers are based on 16S rRNA sequences, their cardiovascular diseases, neurodegenerative diseases, and cancer coverage is currently uncertain in the highly diverse human gut (He and Chen, 2013; Wada et al., 2013; Bilal et al., 2014). ecosystem. Isoflavones-mediated effects appear to be driven by their The aim of the present work was to develop a qPCR method hormonal (Yuan et al., 2007), antioxidant (Arora et al., 1998), and capable of identifying and quantifying equol-producing bacteria enzyme-inhibitory (Crozier et al., 2009) activities. by targeting functional genes involved in the synthesis of this In soy, isoflavones are mostly found as glycoside conjugates compound. This methodology would provide a new tool for (daidzin, genistin, and glycitin) (Franke et al., 2014). Isoflavones evaluating strategies aimed to increase the endogenous formation are more bioavailable after their deglycosylatation by cellular of equol. The specificity and sensitivity of the method was enzymes or enzymes belonging to certain gut bacteria (Franke tested using pure cultures of equol-producing and non-equol- et al., 2014). The aglycone moieties (daidzein, genistein, and producing strains of intestinal bacterial species. The method was glycitein) of isoflavone glycosides are released via the action then tested using human faecal samples, and the slurry cultures of cellular and bacterial β-glucosidases (Islam et al., 2014). derived from them, provided by equol-producing and non- Aglycones are further metabolized into either compounds of equol-producing women. Isoflavones and their metabolites were greater biological activity or inactive molecules (Clavel and quantified by ultra-high performance liquid chromatography Mapesa, 2013). Equol, produced from daidzein, has the strongest (UHPLC). oestrogenic and antioxidant activity of all isoflavone metabolites (Setchell and Clerici, 2010; Franke et al., 2014). All the animal species tested to date (including cows, pigs, sheep, chickens, mice, MATERIALS AND METHODS and rats) produce equol in response to soy (and thus daidzein) intake (Setchell and Clerici, 2010). However, only 30–60% of Human Intervention Study humans do so, and it may be only these who fully benefit from This study was approved by the Bioethics Committee of CSIC soy and/or isoflavone consumption (Franke et al., 2014). (Consejo Superior de Investigaciones Científicas) and by the The human gut microbiota harbours a complex and Regional Ethics Committee for Clinical Research (Servicio de dynamic population of microorganisms, which is dominated Salud del Principado de Asturias, Spain). The selection of by nutritionally-fastidious, strict anaerobic bacteria that are donors and later sampling was performed following standardized extremely sensitive to ambient oxygen (Thursby and Juge, 2017). protocols recommended by the above committees. All subjects Indeed, growth in culture of many of the bacterial species gave written informed consent in accordance with the declaration forming the gut microbiota has, until recently, been considered of Helsinki. The faecal samples analysed in this work, or utilized impossible (Browne et al., 2016). Therefore, culture-independent as inoculants for the faecal cultures, had been collected in techniques are considered more suitable than the traditional a previous intervention study of menopausal women under methods for identifying and quantifying the components of the treatment with a soy isoflavone concentrate (Guadamuro et al., = intestinal microbial populations (Delgado et al., 2013; Kim et al., 2015). In short, participants (n 18; age range 48–61 years, 2015). Among the different, culture-independent, molecular mean 52.6 years; body weight range 52–73 kilo; average 65.3) techniques available, real-time quantitative PCR (qPCR) has consumed for 6 months one tablet a day containing 80 mg of become highly regarded as a specific detector and quantifier isoflavones containing genistin/daidzin in the range of 55–72% of microorganisms in complex microbial samples, including (Fisiogen; Zambon). Faeces were taken a three time points: before samples from the human gut (Furet et al., 2009; Liszt et al., 2009; the start of the intervention (time 0), and at 1, 3, and 6 months of Tuomisto et al., 2013; Ruengsomwong et al., 2014). treatment. Freshly voided stools were collected in sterile plastic Equol biosynthesis from daidzein seems to take place containers and transported to the laboratory, where they were − ◦ through the consecutive action of three conserved reductases via kept frozen at 80 C until analysis. dihydrodaidzein and tetrahydrodaidzein intermediates (Shimada et al., 2010, 2011; Schröder et al., 2013). Though far from Bacteria and Culture Conditions complete, our knowledge of the microorganisms that produce Table 1 shows the bacteria used in the present work. Strains equol from daidzein, and of the biochemical pathways involved, of most species were obtained from the Leibniz Institut- is growing (Yuan et al., 2007; Setchell and Clerici, 2010). Deutsche Sammlung von Mikroorganismen und Zellkulturen In the last decade, a number of bacterial strains capable of (DSMZ) collection. Intestinal species were cultured in either producing equol have been identified from human and animal Gifu Anaerobic Medium (GAM) broth (Nissui Pharmaceuticals, sources (Wang et al., 2005; Uchiyama et al., 2007; Maruo Tokyo, Japan) supplemented with 0.5% arginine (Merck, et al., 2008; Yokoyama and Suzuki, 2008; Yu et al., 2008; Darmstad, Gemany) (GAM-Arg), or Reinforced Clostridium Tsuji et al., 2010). Nearly all those isolated so far fall into the Medium (RCM) (Merck), at 37◦C in a Mac500 anaerobic family Coriobacteriaceae (Clavel et al., 2014), which includes a chamber (Down Whitley Scientific, West Yorkshire, UK) under series of newly described, nutritionally-fastidious species that are anoxic atmospheric conditions (10% H2, 10% CO2, and 80% N2).

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TABLE 1 | Bacterial strains and oligonucleotide primers utilized in this work.

Strain/item Phenotype/sequence Origin/target (position) Source/reference

Adlercreutzia equolifaciens DSM 19450T Equol-producer Human faeces DSMZ Asaccharobacter celatus DSM 18785T Equol-producer Rat caecum DSMZ Enterorhabdus mucosicola DSM 19490T Equol-producer Ileal mucosa DSMZ Slackia equolifaciens DSM 24851T Equol-producer Human faeces DSMZ Slackia isoflavoniconvertens DSM 22006T Equol-producer Human faeces DSMZ Bacteroides fragilis DSM 2151T Equol non-producer Appendix abscess DSMZ Bacteroides thetaiotaomicron DSM 2079T Equol non-producer Human faeces DSMZ Bifidobacterium longum H66 Equol non-producer Human faeces Laboratory collection Blautia coccoides DSM 935T Equol non-producer Mouse faeces DSMZ Blautia producta DSM 2950T Equol non-producer Human septicemia DSMZ Blautia obeum DSM 25238T Equol non-producer Human faeces DSMZ Collinsella intestinalis DSM 13280T Equol non-producer Human faeces DSMZ Escherichia coli A-15 Equol non-producer Dairy biofilm Laboratory collection Faecalibacterium prausnitzii DSM 17677 Equol non-producer Human faeces DSMZ Lactobacillus rhamnosus E41 Equol non-producer Human faeces Laboratory collection Prevotella copri DSM 18205T Equol non-producer Human faeces DSMZ Oligonucleotide primers (5′–3′) tdr.qPCR-F RTYAACGGCRAYATGCAGGT tdr (1279–1298)a This work tdr.qPCR-R GGMAYYTCCATGTTGTAGGA tdr (1372–1391)a This work ddr.qPCR-F CTCGAYCTSGTSTACAACGT ddr (421–440)a This work ddr.qPCR-R GARTTGCAGCGRATKCCGAA ddr (607–626)a This work dzr.qPCR-F GAAGCTTGATATGGACGACT dzr (669–688)a This work dzr.qPCR-R GGAATATGCACCTGTTCCT dzr (854–872)a This work TBA-F CGGCAACGAGCGCAACCC 16S rRNA gene Denman and McSweeney, 2006 TBA-R CCATTGTAGCACGTGTGTAGCC 16S rRNA gene Denman and McSweeney, 2006

(T )Type strain. DSMZ, Leibniz Institut-Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany. aAccording to the numbering of genes in S. isoflavoniconvertens DSM 22006T (GenBank accession number JQ358709).

Escherichia coli, however, was grown in Luria-Bertani (LB) broth USA), culture supernatants were used directly (i.e., without in aerobiosis at 37◦C with shaking. purification or any extraction step) in UHPLC analysis. Cultures were analysed in duplicate. Quantification was performed Faecal Cultures against calibration curves prepared using commercially available Ten-fold faecal dilutions were prepared by homogenizing 1 g standards. of faeces in 9 ml of pre-reduced phosphate buffer saline (PBS) in an anaerobic atmosphere (as above). A 10% (v/v) aliquot of DNA Extraction from Bacteria, Faecal the resulting faecal slurry was used to inoculate GAM-Arg to Samples, and Faecal Cultures which the isoflavones daidzein or genistein (Toronto Research Total DNA from both control bacteria and microorganisms from Chemical, Toronto, Canada) were added at a concentration of faeces and faecal cultures was extracted following the procedure 100 µM. Faecal cultures were then incubated in open tubes of Zoetendal et al. (2006) using the QIAamp DNA Stool ◦ under anaerobic conditions at 37 C for 24 h. They were then Minikit (Qiagen, Hilden, Germany), with minor modifications as centrifuged at 13,500 rpm for 5 min, and the supernatants and reported by Guadamuro et al. (2015). DNA was finally eluted with pellets independently collected for isoflavone metabolite analysis 150 µL sterile molecular biology grade water (Sigma-Aldrich, St. and DNA extraction, respectively. Louis, CA., USA) and stored at −20◦C until use in real-time (qPCR) amplifications. Detection and Quantification of Isoflavone Metabolites Design of Primers Targeting Genes Daidzein and genistein, and their metabolites dihydrodaidzein, Involved in Equol Production dihydrogenistein, and equol, were measured in faecal cultures Genome sequences of equol-producing bacteria deposited in by a UHPLC procedure based on the method for determining the NCBI database (http://www.ncbi.nlm.nih.gov/genome/) isoflavones in urine (Redruello et al., 2015). After filtering were downloaded. Sequences of the genes encoding reductases through a 0.2 µm PTFE membrane (VWR, Radnor, PA, identified in equol-associated gene clusters [tetrahydrodaidzein

Frontiers in Microbiology | www.frontiersin.org June 2017 | Volume 8 | Article 1155 41 CAPÍTULO 1 Vázquez et al. qPCR Quantification of Equol-Producing Bacteria reductase (tdr), dihydrodaidzein reductase (ddr) and daidzein calculated from the slope of the standard curve for each primer reductase (dzr)] were then aligned using Clustal Omega software set using the formula E = 10−1/slope. Positive amplification was (http://www.ebi.ac.uk/Tools/msa/clustalo/) (Supplementary deemed to have occurred when a Ct value of ≤30 was recorded; Figure 1). Degenerated oligonucleotide primers were manually this corresponds to a total bacteria detection limit of <102 cfu/ml designed based on the conserved regions of the genes (Table 1). (as determined by amplification with the 16S rRNA-encoding The efficacy and specificity of the primers were evaluated using gene primers mentioned above; Table 1). DNA from equol-producing and non-producing intestinal bacteria as a template (Table 1). Statistical Analysis Real-Time qPCR qPCR data were analysed using free R software (http://www.r- project.org). The Shapiro-Wilk test was used to check for the Real-time qPCR was performed using a 7,500 Fast Real- normal distribution of the data. The non-parametric Spearman time PCR System running software version 2.0.4 (Applied rank correlation test was used to examine the relationship Biosystems, Foster City, CA., USA). Amplification and detection between the copy numbers of tdr and ddr, and between gene copy were performed in 96-well optical plates (Applied Biosystems) number and equol production. The Wilcoxon signed-rank test with SYBR-Green (Applied Biosystems). All amplifications were was used to determine whether the copy number of tdr or ddr performed in triplicate in a final volume of 20 µL containing genes differed over isoflavone treatment (0, 1, 3, and 6 months). 10 µL of a 2xSYBR Green PCR Master Mix including ROX The same test was used to correlate gene copy numbers and equol as a passive reference (Applied Biosystems), 900 nM of each production. Significance was set at P < 0.05. primer, and 2 µL of template DNA (5–10 ng). For amplification, the standard protocol of the 7,500 thermocycler (Applied Biosystems) was followed, i.e., an initial cycle at 95◦C for 10 min, RESULTS followed by 40 cycles at 95◦C for 15 s, and 1 min at 60◦C. To check for specificity, melting curve (Tm) analysis was performed, Alignment of the database-available reductase-encoding genes increasing the temperature from 60 to 95◦C at a rate of 0.2◦C showed the tdr and ddr sequences from the different species per second with the continuous monitoring of fluorescence. and strains shared sufficient nucleotide identity (71 and 78%, Equol-producing bacteria in faecal samples were enumerated respectively; Supplementary Figure 2) to allow the design of using standard curves for genes coding for reductases in equol- “universal equol-related” primers (Table 1). However, the dzr producing strains (Table 2). These curves were prepared using genes were so divergent (36% nucleotide identity only) that no 10-fold serial dilutions of DNA extracted from cultures of equol- primers could be designed that could detect all sequences. In an producing strains of known size (determined in GAM-Arg agar attempt to obtain at least some partial information on this gene, a plates at 37◦C after 72 h under anaerobic conditions). The pair of primers (Table 1) based on the sequence of the dzr genes absence of PCR inhibitors in negative samples was ruled out by from the two Slackia strains available -S. isoflavoniconvertens amplifying prokaryotic 16S rRNA gene sequences using universal DSM 22006T (Schröder et al., 2013) and Slackia spp. NATTS primers (Table 1). The efficiency of the equol-related primers was (Tsuji et al., 2012)—were synthesized.

TABLE 2 | Temperature of melting, efficiency and regression equation obtained for the amplification of equol-associated reductase genes with the primers designed in this study and using as a template purified DNA from equol-producing bacteria.

Equol-producing organism/target gene Melting temperature Efficiency (R2) Regression equation

TETRAHYDRODAIDZEIN REDUCTASE (tdr) Gene Adlercreutzia equolifaciens DSM 19450T 83.85 ± 0.09 0.992 y = −0.2907x + 11.266 Asaccharobacter celatus DSM 18785T 83.61 ± 0.17 0.992 y = −0.3007x + 12.190 Enterorhabdus mucosicola DSM 19490T 82.97 ± 0.39 0.996 y = −0.2917x + 11.172 Slackia equolifaciens DSM 24851T 84.05 ± 0.16 0.993 y = −0.3170x + 11.200 Slackia isoflavoniconvertens DSM 22006T 83.39 ± 0.19 0.994 y = −0.3040x + 11.110 DIHYDRODAIDZEIN REDUCTASE (ddr) Gene A. equolifaciens DSM 19450T 88.98 ± 0.23 0.974 y = −0.2662x + 11.296 A. celatus DSM 18785T 88.32 ± 0.09 0.994 y = −0.2729x + 11.780 E. mucosicola DSM 19490T 88.47 ± 0.23 0.988 y = −0.2727x + 11.252 S. equolifaciens DSM 24851T 88.29 ± 0.14 0.992 y = −0.2716x + 10.905 S. isoflavoniconvertens DSM 22006T 88.34 ± 0.20 0.998 y = −0.2946x + 10.821 DAIDZEIN REDUCTASE (dzr) Gene S. equolifaciens DSM 24851 - - - S. isoflavoniconvertens DSM 22006T 85.91 ± 0.36 0.999 y = −0.3065x + 10.998 y, values of log (cfu/ml); x, values of Ct. −,primers gave no amplification when DNA from S. equolifaciens was used as a template.

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The specificity of the primers was experimentally tested Table 2 summarises the key parameters of the amplification against purified DNA from pure cultures of strains belonging reactions using the three pairs of primers developed. Figure 1 to 16 representative bacterial species from the human gut shows the standard curves for the qPCR detection of tdr, ddr and (Table 1). Positive and negative qPCR assays corroborated in dzr, prepared using serial dilutions of DNA containing known silico predictions. Amplification was only obtained when DNA numbers of equol-producing microorganisms. Linear regressions from equol producing strains was used as a template (Table 1). were obtained by plotting the cycle threshold (Ct) values against

FIGURE 1 | Standard curves of qPCR for the tetrahydrodaidzein reductase (tdr) and the dihydrodaidzein reductase (ddr) target genes using serial dilutions of DNA from known amounts of cells of equol-producing microorganisms. Linear regression was obtained plotting the cycle threshold (Ct) values vs. the log10 of the counting results (in cfu/ml). The equation and R2 value of the regression lines are indicated in each panel.

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the log10 enumeration values for the equol producing strains (in TABLE 3 | Cycle threshold (Ct) values obtained in faecal samples for cfu/ml). The detection limit of the qPCR assay was determined tetrahydrodaidzein reductase (tdr) and dihydrodaidzein reductase (ddr) genes and using genomic DNA from these cultures, assuming a genome size absolute abundance of equol producing bacteria in the real-time PCR assay developed in this study. of about 3.0 fg of DNA per cell (Rodríguez-Lázaro et al., 2004). Three independent experimental assessments of the detection Women Sample qPCR amplification of total microbial DNA from limit of the qPCR reaction determined a sensitivity of 1–10 equol- (time)a faeces producing bacteria or genome equivalents, giving a detection Ct Log Ct Log 2 10 10 limit of about 10 cfu/g of faeces. As shown in Table 2, the (tdr) (cfu/ml) ±SD (ddr) (cfu/ml) ± SD efficiency of the primer pair for amplifying the dzr gene was adequate when DNA from S. isoflavoniconvertens was used as a EQUOL PRODUCERS template, but not when the DNA came from S. equolifaciens. This W3 0 25.95 ± 0.01 3.57 ± 0.54 25.45 ± 0.05 4.17 ± 0.58 pair of primers was, therefore, no longer used. 1 23.75 ± 0.70 4.23 ± 0.53 25.00 ± 0.40 4.30 ± 0.58 The primers for tdr and ddr were then used to explore DNA 3 24.98 ± 0.18 3.86 ± 0.54 24.90 ± 0.04 4.32 ± 0.58 purified from the faecal samples of the 18 isoflavone-treated 6 26.24 ± 0.04 3.48 ± 0.54 25.91 ± 0.01 4.05 ± 0.58 menopausal women before starting isoflavone intake (time 0) and W8 0 22.53 ± 0.14 4.60 ± 0.52 22.25 ± 0.03 5.06 ± 0.55 after 1, 3, and 6 months of treatment. Three of these women (W3, 1 22.86 ± 0.22 4.50 ± 0.52 22.53 ± 0.19 4.98 ± 0.55 W8, and W18) had previously been shown to have an equol- 3 22.53 ± 0.22 4.60 ± 0.52 21.67 ± 0.04 5.22 ± 0.55 producing phenotype based on their urine equol/creatinine ratios 6 22.53 ± 0.44 4.60 ± 0.52 21.43 ± 0.18 5.28 ± 0.55 of >5.0 (Guadamuro et al., 2015). Table 3 shows the results W18 0 28.17 ± 0.57 2.90 ± 0.56 - <2 of the amplification, based on the Ct value, and the calculated 1 24.80 ± 0.22 3.92 ± 0.54 absolute abundance of equol-producing organisms. Negative 3 26.02 ± 0.25 3.55 ± 0.54 amplification (absence of target genes) was accepted when the Ct 6 26.11 ± 0.22 3.52 ± 0.54 was >30. As expected, tdr was detected in all samples provided EQUOL NON-PRODUCERS by the equol-producing women. However, the ddr gene was W1 0 - <2 - <2 identified in the faeces of only two (W3 and W8) of the three 1 equol producers, while no amplification was obtained for this 3 gene when used as template DNA for examining the faeces 6 and faecal slurry cultures of subject W18. Changes in the Ct W2 0 - <2 - <2 values of samples from each of the women were observed over 1 the isoflavone treatment period, but with no particular trend 3 apparent. Indeed, Wilcoxon analysis of the values at different 6 time points revealed no significant differences (Supplementary W4 0 - <2 - <2 Figure 3). It is noteworthy that when positive amplifications 1 were detected for tdr and ddr, equivalent copy numbers were 3 always observed (Spearman coefficient 0.918). As a pattern 6 of the Tm curve in qPCR amplicons correlates with specific W5 0 - <2 - <2 nucleotide sequences of amplicons, analysis of the Tm curves 1 provides information on the number of sequences amplified in 3 the reaction and their relationships. In this sense, analysis of the 6 Tm curves (Figure 2) showed some amplicons (those of the tdr W6 0 - <2 - <2 genes from samples provided by subjects W3 and W8, and that 1 of ddr from subject W3) to have Tm patterns similar to those 3 of equol-producing control bacteria (Table 2). In contrast, the 6 Tm of other amplicons (tdr from W18 and ddr from W8) were W7 0 22.49 ± 0.08 4.61 ± 0.52 23.42 ± 0.05 4.73 ± 0.56 rather different to those of the positive strains. Moreover, the ddr 1 23.40 ± 0.02 4.34 ± 0.53 24.30 ± 0.13 4.49 ± 0.57 amplicon from W8 showed two separate peaks, indicating that 3 23.57 ± 0.13 4.29 ± 0.53 24.60 ± 0.01 4.41 ± 0.57 two DNA fragments with different sequence are being amplified. 6 28.01 ± 0.51 2.95 ± 0.56 27.56 ± 0.08 3.59 ± 0.57 It was also surprising to find amplicons of both tdr and W9 0 - <2 - <2 ddr when examining the samples of two non-equol-producing 1 women (W7 and W15) (Table 3). To confirm that the phenotypic 3 results of equol production (whether positive and negative) 6 had been maintained, and to gain further insights into the W10 0 - <2 - <2 metabolism of soy isoflavones, faecal slurries from selected faecal 1 samples provided by equol-producing and non-equol-producing 3 women (including those in which tdr and ddr were detected) 6 were inoculated into GAM-Arg medium with no isoflavones (control) or with either daidzein (DZEN) or genistein (GTEN), (Continued)

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TABLE 3 | Continued dihydrodaidzein) were recovered from these cultures. Variable amounts of genistein and its derived metabolite dihydrogenistein Women Sample qPCR amplification of total microbial DNA from (time)a faeces were also recovered from cultures when this isoflavone was added. The exception was the faecal culture from W8, in which no Ct Log10 Ct Log10 genistein (detection limit 15.17 nM [Redruello et al., 2015]) and ± ± (tdr) (cfu/ml) SD (ddr) (cfu/ml) SD only a small quantity of dihydrogenistein (0.41 µM), was scored.

W11 0 - <2 - <2 The qPCR results obtained for the faecal cultures matched 1 those obtained with DNA isolated from faeces. The data agreed 3 well both qualitatively and quantitatively, with the Ct values of the corresponding faeces and faecal culture samples following the 6 same trend. The tdr gene was shown to be present in cultures W12 0 - <2 - <2 inoculated with faecal material from all three equol-producing 1 women (W3, W8, and W18), while ddr was only detected in the 3 cultures from subjects W3 and W8. Once again, both tdr and 6 ddr were identified in cultures from the non-equol-producing W13 0 - <2 - <2 subjects W7 and W15. 1 3 6 DISCUSSION W14 0 - <2 - <2 1 Conventional means of identifying and quantifying 3 microorganisms in complex ecosystems, such as those in 6 the gastrointestinal tract, are laborious, time consuming, and W15 0 24.16 ± 0.12 4.11 ± 0.53 25.77 ± 0.15 4.09 ± 0.52 only recover the cultivable part of their populations (Qin et al., 1 23.22 ± 0.08 4.39 ± 0.53 26.28 ± 0.25 3.94 ± 0.52 2010; Browne et al., 2016). Molecular, culture-independent 3 23.91 ± 0.16 4.18 ± 0.53 27.18 ± 0.14 3.70 ± 0.53 microbial techniques, such as qPCR, are therefore essential 6 25.78 ± 0.12 3.62 ± 0.54 26.64 ± 0.02 3.84 ± 0.53 if gut-dwelling microorganisms are to be reliably identified W16 0 - <2 - <2 and quantified. This is particularly important when tracking 1 microorganisms involved in intestinal functionality, such as 3 those that produce equol (Clavel et al., 2014). Strain-specific 6 oligonucleotide primers based on 16S rRNA gene sequences W17 0 - <2 - <2 for the equol-producing Slackia spp. NATTS have already been 1 reported (Tsuji et al., 2010; Sugiyama et al., 2014). 16S rRNA 3 gene-based primers targeting Coriobacteriaceae species have also 6 been developed (Harmsen et al., 2000; Thorasin et al., 2015; Cho et al., 2016). However, as the microbial typing of the human gut −, qPCR negative (Ct > 30.00). aSamples were taken before the start of 0 and at 1, 3, and 6 months during isoflavone microbiota is not yet complete (Harmsen and de Goffau, 2016), treatment. the coverage of the current coriobacterial primers is uncertain. Further, whether equol-production is a phylogeny-related trait and incubated for 24 h at 37◦C under anaerobic conditions. (species-specific) or an acquired property (strain-specific) has DNA isolated from the faecal cultures was then subjected yet to be determined (Clavel and Mapesa, 2013). Thus, primers to qPCR analysis for the detection and quantification of tdr targeting functional, single-copy genes, such as those involved in and ddr under the same conditions as above. In addition, equol biosynthesis, are preferable. isoflavones and their metabolites, including daidzein, genistein, The present work reports a qPCR assay involving dihydrodaidzein, dihydrogenistein, and equol, were measured oligonucleotide primers based on conserved sequences of in the faecal cultures by UHPLC. Table 4 shows the results reductase-encoding genes implicated in the synthesis of equol, obtained. No isoflavones or their metabolites were ever detected plus the use of SYBR Green as a dye, for the detection and in the control cultures without added isoflavones. When daidzein quantification of equol-producing bacteria. The main advantage and genistein were added to the faecal slurry cultures they of using SYBR Green instead of molecular probes is its lower were recovered from the uninoculated samples in varying cost and a reduced need for optimisation (Inglis and Kalischuk, amounts (67–81% of the added amounts). However, the added 2004). The specific binding of SYBR Green to any double daidzein completely disappeared (converted into equol) when stranded nucleic acid allows the detection of non-specific the medium containing this isoflavone was inoculated with S. and/or multiple amplifications by Tm curve analysis. The isoflavoniconvertens DSM 22006. Equol in the faecal cultures proposed qPCR assay clearly distinguished target species from was only present in those provided by the equol-producing all non-target species analysed belonging to the same ecosystem, women. However, the transformation of daidzein into equol was thus demonstrating its specificity. It also showed excellent never complete, and variable amounts of daidzein (and usually quantification characteristics in terms of both linear dynamic

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FIGURE 2 | Melting curves of qPCR amplicons with the primers designed in this study targeting the tetrahydrodaidzein reductase (tdr) gene (A) and dihydrodaidzein reductase (ddr) (B) gene using as a template total microbial DNA from cultures containing daidzein inoculated at 1% with feces from equol producing women W3 (in green), W8 (in blue), and W18 (in red) and no-equol producing women W7 (in orange) and W15 (in pink). Note that the ddr gene gave no amplification in cultures derived from samples of woman W18 (B). range and relative accuracy. In addition, it performed equally peaks observed in the Tm of samples from subject W8 -although, well with purified DNA from equol-producing bacteria and non-specific amplification would also give rise to the same total microbial DNA from faeces and faecal slurry cultures. results. Sequencing and analysis of amplicons obtained through Unfortunately, since only limited information on the microbial conventional PCR using primers targeting other conserved types involved in the synthesis of equol is available, the regions of both ddr and tdr genes further support the presence identification and quantification of equol producers by qPCR of not yet reported genes in some faecal samples (data not technique developed in this work might be compromised. This shown). An unrelated ddr gene might also be carried by subject could result in the underestimation of the amount of DNA W18, for whose samples no amplification of this gene was present (and thus the number of equol-producing bacteria), or ever recorded. Altogether, these observations suggest that equol- even in amplification failure. Sequences of genes from newly associated genes (and thus equol-producing bacteria) unrelated discovered equol-producing bacteria could be incorporated into to those reported in the literature (Jin et al., 2010; Shimada et al., the design of “universal”, “group-specific,” or “species-specific” 2010, 2012; Tsuji et al., 2012; Schröder et al., 2013; Tho et al., primer pairs to improve the sensitivity, coverage and accuracy of 2013) might be present in the gut of the subjects of this study. the proposed assay. It is interesting to note that, when positive Among the inconsistencies regarding the detection of genes amplification of tdr and ddr was observed, equivalent copy and equol production, it is noteworthy the positive amplification numbers of these two genes were recorded, suggesting they are of both tdr and ddr in DNA from faeces and slurry cultures located in the same genetic element (such as in the chromosome of two non-equol-producing women. The presence of non- in an operon-like structure for S. isoflavoniconvertens; Schröder functional, yet amplifiable genes, the conversion of daidzein et al., 2013; Tho et al., 2013). The fact that the copy number into downstream metabolites other than equol, and the presence of the genes did not increase significantly over the course of of homologous genes encoding enzymes without activity subjects’ treatment suggests that equol-producing bacteria are over daidzein, could account for this apparent contradiction. not being positively selected by the short-term isoflavone intake However, the addition of equol to faecal cultures from subjects assessed in this study. The slight increase in Coriobacteriaceae W7 and W15, excluded the possibility of any further metabolism members seen during isoflavone consumption (Nakatsu et al., of this compound (data not shown). The presence of related 2014; Guadamuro et al., 2015) might be a consequence of the genes might be supported by the recovery of a large proportion inhibition of dominant intestinal bacterial populations by these of the genistein in the faecal cultures of these two women compounds or their metabolites. as dihydrogenistein (Table 4). From a chemical point of view, Analysis of the Tm amplification curves suggests the presence daidzein and genistein are highly similar molecules (del Rio et al., in the faecal samples of some women of gene sequences identical 2013), which suggests that enzymes acting on these compounds or very similar to those of the equol-producing bacterial species might show structural and functional similarities. Indeed some used as a control. However, in other subjects, Tm values different enzymes, as has been demonstrated for the reductases of to those of the controls were also observed, indicating the S. isoflavoniconvertens, act on both daidzein and genistein presence of genes in their faeces with nucleotide differences, aglycones (Matthies et al., 2012). Therefore, the presence of genes which suggests the involvement of unrelated taxa in equol coding for enzymes acting in genistein only but with enough formation in the faeces of these individuals. The presence of nucleotide identity to be amplified with the primers of this study two equol-producing species might be responsible for the two without equol production is plausible. If this were the case, it

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TABLE 4 | Cycle threshold (Ct) values obtained by qPCR for tetrahydrodaidzein reductase (tdr) and dihydrodaidzein reductase (ddr) genes and isoflavone metabolites in the faecal slurry cultures.

Faecal sample GAM-Arg witha qPCR amplification from faecal cultures Isoflavone metabolites in faecal cultures (in µM)

Ct (tdr) Ct (ddr) Daidzein Dihydrodaidzein Genistein Dihydrogenistein Equol

EQUOL PRODUCERS W3.3 Control 25.28 ± 0.07 26.07 ± 0.14 - - - - - DZEN 23.57 ± 0.17 24.60 ± 0.08 43.03 28.28 - - 10.99 GTEN 26.30 ± 0.02 26.84 ± 0.04 - - 24.42 22.36 - W8.1 Control 25.34 ± 0.38 23.47 ± 0.17 - - - - - DZEN 22.32 ± 0.31 21.10 ± 0.07 49.45 0.87 - - 10.69 GTEN 21.65 ± 0.43 20.78 ± 0.17 - - - 0.41 0.15 W18.1 Control 28.67 ± 0.55 ------DZEN 29.56 ± 0.43 - 54.36 2.27 - - 11.01 GTEN 28.95 ± 0.20 - - - 57.46 2.15 - EQUOL NON-PRODUCERS W1.3 Controla ------DZEN - - 69.54 - - - - GTEN - - - - 16.30 0.94 - W5.3 Control ------DZEN - - 42.22 37.80 - - - GTEN - - - - 15.40 23.95 - W7.3 Control 22.52 ± 0.15 23.24 ± 0.16 - - - - - DZEN 25.61 ± 0.07 26.35 ± 0.04 80.32 - - - - GTEN 24.86 ± 0.01 25.70 ± 0.04 - - 2.48 83.17 - W15.3 Control 25.81 ± 0.04 26.90 ± 0.02 - - - - - DZEN 25.41 ± 0.09 26.26 ± 0.10 72.03 1.14 - - - GTEN 25.62 ± 0.11 26.54 ± 0.04 - - 5.81 50.80 - W17.1 Control ------DZEN - - 72.37 9.19 - - - GTEN - - 47.77 5.98 - CULTURE CONTROLS S. isoflavb Control nd nd - - - - - DZEN nd nd - - - - 100.00 GTEN nd nd - - 45.76 6.69 0.79 GAM-Argc Control nd nd - - - - - DZEN nd nd 81.18 - - - - GTEN nd nd - - 67.00 - -

-, qPCR negative (Ct > 30.00) or isoflavone metabolite under the limit of detection; nd, not done. aThe medium used for the faecal cultures (GAM-Arg) contained either daidzein (DZEN), genistein (GTEN), or no isoflavones (Control). bS. Isoflav, culture of Slackia isoflavoniconvertens DSM 22006 in GAM-Arg. cGAM-Arg, uninoculated culture medium incubated under the same conditions. would result in DNA (and thus the number of equol-producing (urine) and culture supernatants by chromatographic methods bacteria) overestimation. would help tracking equol-producing populations in the gut. In conclusion, this work reports a highly specific, sensitive Monitoring equol-producing microorganisms in the human and reliable qPCR assay for the detection and quantification of gut could provide a means of evaluating strategies aimed at equol-producing bacteria in microbiologically complex samples, increasing the endogenous formation of this compound. The including human-derived faecal samples and faecal cultures. biological significance of the presence/absence of tdr and ddr Combining the qPCR technique described here with the genes in isoflavone metabolism and equol production is currently detection and quantification of equol in biological fluids under study.

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AUTHOR CONTRIBUTIONS contracts from the FPI Program and from MINECO (BES-2015-072285 and BES-2012-062502, respectively). BM and AF conceived the study. LV and LG were involved AF was supported by a JAE-Doc Program contract from in the experimental determinations. FG engaged participants the CSIC. The skillful assistance of Begoña Redruello, and provided samples. BM provided materials and resources. Servicios Científico-Técnicos of IPLA-CSIC for the analysis AF drafted the manuscript. BM made a critical revision of the of isoflavone metabolites in fecal cultures is greatly manuscript. All authors reviewed and approved the final version. acknowledged.

ACKNOWLEDGMENTS SUPPLEMENTARY MATERIAL

This study was supported by projects from the Spanish The Supplementary Material for this article can be found Ministry of Economy and Competitiveness (MINECO) online at: http://journal.frontiersin.org/article/10.3389/fmicb. (AGL2014-57820-R) and the Principality of Asturias 2017.01155/full#supplementary-material (GRUPIN14-137). LV and LG were supported by research

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Microbiol. Biotechnol. 24, 1026–1033. doi: 10.4014/jmb.1310.10043 21, 217–220. doi: 10.11209/jim.21.217 Schröder, C., Matthies, A., Engst, W., Blaut, M., and Braune, A. (2013). Wada, K., Nakamura, K., Tamai, Y., Tsuji, M., Kawachi, T., Hori, A., et al. (2013). Identification and expression of genes involved in the conversion of daidzein Soy isoflavone intake and breast cancer risk in Japan: from the Takayama study. and genistein by the equol-forming bacterium Slackia isoflavoniconvertens. Int. J. Cancer 133, 952–960. doi: 10.1002/ijc.28088 Appl. Environ. Microbiol. 79, 3494–3502. doi: 10.1128/AEM.03693-12 Wang, X. L., Hur, H. G., Lee, J. H., Kim, K. T., and Kim, S. I. (2005). Setchell, K. D., and Clerici, C. (2010). Equol: history, chemistry, and formation. J. Enantioselective synthesis of S-equol from dihydrodaidzein by a newly isolated Nutr. 140, 1355S–1362S. doi: 10.3945/jn.109.119776 anaerobic human intestinal bacterium. Appl. Environ. Microbiol. 71, 214–219. Shimada, Y., Takahashi, M., Miyazawa, N., Abiru, Y., Uchiyama, S., and Hishigaki, doi: 10.1128/AEM.71.1.214-219.2005 H. (2012). Identification of a novel dihydrodaidzein racemase essential for Yokoyama, S., and Suzuki, T. (2008). Isolation and characterization of a novel biosynthesis of equol from daidzein in Lactococcus sp. strain 20-92. Appl. equol-producing bacterium from human feces. Biosci. Biotechnol. Biochem. 72, Environ. Microbiol. 78, 4902–4907. doi: 10.1128/AEM.00410-12 2660–2666. doi: 10.1271/bbb.80329 Shimada, Y., Takahashi, M., Miyazawa, N., Ohtani, T., Abiru, Y., Uchiyama, Yu, Z. T., Yao, W., and Zhu, W. Y. (2008). Isolation and identification of equol- S., et al. (2011). Identification of two novel reductases involved in equol producing bacterial strains from cultures of pig faeces. FEMS Microbiol. Lett. biosynthesis in Lactococcus strain 20-92. J. Mol. Microbiol. Biotechnol. 2, 282, 73–80. doi: 10.1111/j.1574-6968.2008.01108.x 160–172. doi: 10.1159/000335049 Yuan, J.-P., Wang, J.-H., and Liu, X. (2007). Metabolism of dietary soy isoflavones Shimada, Y., Yasuda, S., Takahashi, M., Hayashi, T., Miyazawa, N., Sato, I., et al. to equol by human intestinal microbiota. Implications for healthMol. Nutr. (2010). Cloning and expression of a novel NADP(H)-dependent daidzein Food Res. 51, 765–781. doi: 10.1002/mnfr.200600262 reductase, an enzyme involved in the metabolism of daidzein, from equol- Zoetendal, E. G., Heilig, H. G. H. J., Klaassens, E. S., Booijink, C. C. G. M., producing Lactococcus strain 20-92. Appl. Environ. Microbiol. 76, 5892–5901. Kleerebezem, M., Smidt, H., et al. (2006). Isolation of DNA from bacterial doi: 10.1128/AEM.01101-10 samples of the human gastrointestinal tract. Nat. Protocols 1, 870–873. Sugiyama, Y., Nagata, Y., Fukuta, F., Takayanagi, A., Masumori, N., Tsukamoto, doi: 10.1038/nprot.2006.142 T., et al. (2014). Counts of Slackia sp. strain NATTS in intestinal flora are correlated to serum concentrations of equol both in prostate cancer cases Conflict of Interest Statement: The authors declare that the research was and controls in Japanese men. Asian Pacific J. Cancer Prev. 15, 2693–2697. conducted in the absence of any commercial or financial relationships that could doi: 10.7314/APJCP.2014.15.6.2693 be construed as a potential conflict of interest. Tho, H., Oshima, K., Suzuki, T., Hattori, M., and Morita, H. (2013). Complete genome sequence of the equol-producing bacterium Copyright © 2017 Vázquez, Guadamuro, Giganto, Mayo and Flórez. This is an Adlerkreutzia equolifaciens DSM 19450. Genome Announc. 1:e00742. open-access article distributed under the terms of the Creative Commons Attribution doi: 10.1128/genomeA.00742-13 License (CC BY). The use, distribution or reproduction in other forums is permitted, Thorasin, T., Hoyles, L., and McCartney, A. L. (2015). Dynamics and diversity provided the original author(s) or licensor are credited and that the original of the ‘Atopobium cluster’ in human faecal microbiota, and phenotypic publication in this journal is cited, in accordance with accepted academic practice. characterization of ‘Atopobium cluster’ isolates. Microbiol. 161, 565–579. No use, distribution or reproduction is permitted which does not comply with these doi: 10.1099/mic.0.000016 terms.

Frontiers in Microbiology | www.frontiersin.org June 2017 | Volume 8 | Article 1155 49 CAPÍTULO 1

MATERIAL SUPLEMENTARIO

T P P T T P tdr ddr dzr

ifcA ifcB ifcC ifcD ifcE

Supplementary Figure 1.- Schematic representation of equol gene cluster from Slackia isoflavoniconvertens DSM 22006T (taken from Schröder et al., 2013). All genes within the cluster are induced in the presence of daidzein. Reductase-like genes are marked in green. Tetrahydrodaidzein reductase (tdr), dihydrodaidzein reductase (ddr) genes, and daidzein reductase (dzr) genes are marked in green. Genes ifcA (dihydrodaidzein racemase), ifcB and ifcC (β-subunit and the α-subunit, respectively, of electron-transferring flavoproteins), ifcD (hypothetical protein) and ifcE (NADPH-dependent glutamate synthase β chain-like oxidoreductases) are marked in light pink. Putative promoters (P) and terminators (T) within the operon are also depicted. Approximate position of the primers developed in this study for the different genes is also indicated.

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A 100% 95% 90% 85% 80% 75% 70%

Slackia isoflavoniconvertens (AFV15450) 85%

Slackia spp. NATTS (BAL46928) 80%

Lactococcus garvieae (BAJ72747) 100% Eggerthella spp. (EGYY_15760) 71%

Adlercreutzia equolifaciens (AEQU_2231) 94% Enterorhabdus mucosicola (NZ_KE383895) B 100% 95% 90% 85% 80% 75%

Slackia isoflavoniconvertens (AFV15451) 92%

Slackia spp. NATTS (BAL46928) 78% Lactococcus garvieae (BAJ72748) 100% Eggerthella spp. (EGYY_15750) 81%

Adlercreutzia equolifaciens (AEQU_2230) 95% Enterorhabdus mucosicola (NZ_KE383895)

C 100% 90% 80% 70% 60% 50% 40% 30%

Slackia isoflavoniconvertens (AFV15453) 91%

Slackia spp. NATTS (BAL46928) 55% Eggerthella spp. (EGYY_15730) 72% Adlercreutzia equolifaciens (AEQU_2228) 99% 36% Enterorhabdus mucosicola (NZ_KE383895)

Lactococcus garvieae (BAJ72750)

Supplementary Figure 2.- Homology tree showing the identity percentage between genes involved in equol production described in several equol-producing strains. Panel A, homology of the tetrahydrodaidzein reductase (tdr) genes; Panel B, homology of the dihydrodaidzein reductase (ddr) genes; Panel C, homology of the daidzein reductase (dzr) genes. In parenthesis, GenBank accession numbers of gene sequences from the different microorganisms are indicated.

51 Supplementary Figure 3.- 3.- Figure Supplementary respectively) respectively) for values cultures (Panelscultures C Ct (tdr) Ct (tdr) 24 24 28 28 22 26 28 22 26 C C A Control tdr and 0 0

and Faecal Faecal culture Sampling time time Sampling in Sample Sample time and

the ddr Dzen Dzen D) faecal slurry slurry faecal genes 1 1 . Box diagramma diagramma Box in Gten Gten acl ape al ln tetet Pnl A (Panels treatment along all samples faecal 3 3 of control control of 52

the daidzein (dzen) daidzein and Ct (ddr)

Wilconson test test Wilconson Ct (ddr) 24 24 28 28 22 26 26 28 22

D D B Control 0 0 for Sampling time time Sampling Faecal Faecal culture genistein ( genistein and related samples related Dzen Dzen 1 1 CAPÍTULO gten Gten Gten of 3 3 ) ) and

added the 1

Ct B,

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Article Composition and functionality of faecal microbiota from equol producer and equol non-producer women as assessed by shotgun metagenomics

Short title: Shotgun metagenomics of faeces from equol producers

Abstract: Equol synthesized from daidzein by minority bacterial populations in the human gut is the compound having the strongest estrogenic activity and antioxidant action among the isoflavones and isoflavone-derived metabolites. Thus, the beneficial effects on human health of soy consumption might partially or totally be attributed to equol. Although some intestinal species involved in the formation of equol have been identified, the association between the composition and functionality of the gut microbiota and the equol-producing status remains scarcely investigated. In this study, a shotgun sequencing approach was applied to total microbial DNA from faecal samples in the search for differences and similarities in the microbiota composition and functionality between equol-producing (n=3) and equol non-producing (n=2) women, with a special focus on equol- producing taxa and their equol-associated genes. The microbial diversity between all faecal samples was highly similar at the phylum, genus, and species level, and only a scarce variability regarding relative abundances were detected. Bacteria of the family Eggerthellaceae and, more precisely, to the equol-producing species Adlercreutzia equolifaciens and Eggerthella sp. were found at similar numbers in samples from both equol producers and equol non-producers. Indeed, a correlation between abundance of equol-producing taxa and the equol-producing phenotype was not found. Functional metagenomics analysis showed higher abundance of DNA sequences involved in carbohydrate metabolism and short chain fatty acids synthesis in samples from equol producers. However, metabolic pathways and genes involved in equol production were not identified, even in samples from equol producers. By aligning reductase-encoding genes involved in equol production with the metagenomics data, a single read of a daidzein reductase gene was recognized in a sample from one equol-producing woman. Taxonomic results suggest that the phylogenetic identification might not an accurate approach for detecting and quantifying equol-producing microbes in the gut. Functional analysis could be an alternative, although deeper shotgun sequencing should be reached.

Keywords: Equol, daidzein, microbiota, microbiome, shotgun metagenomics

1. Introduction The consumption of isoflavone-containing foods community and dietary habits, can produce equol (mostly soy and soy-derived products) has (Franke et al., 2014). It is firmly established that the traditionally been linked with positive effects on production of equol requires the presence in the gut human health (for recent reviews see, Messina, 2016; microbiota of specific equol-producing bacteria Zaheer et al., 2017). Epidemiological studies have (Setchell and Cole, 2006; Bolca et al., 2007; Hall et further reinforced the relationship between regular al., 2007; Yoshikata et al., 2019). However, the isoflavone intake and the relief of menopause systematic study of the involvement of intestinal symptoms in women and a reduction of the risk of microbes in isoflavone metabolism and equol cardiovascular and neurodegenerative diseases and synthesis is yet underexplored (Rafii et al., 2015 and certain types of cancer (Bolaños et al., 2010; Wada Mayo et al., 2019). et al., 2013; Bilal et al., 2014). Among isoflavones Currently, only a small number of strains have and their derived metabolites, equol synthesized been identified to be capable of carrying out the from daidzein is the compound having the strongest transformation of daidzein into equol. With the estrogenic action and the highest antioxidant activity exception of a few strains, most equol-producing (Jackson et al., 2011; Kładna et al., 2016), by which bacteria from intestinal sources belong to the family isoflavones and equol are thought to mediate their Eggerthellaceae, and particularly, to the genera claimed health benefits. The production of equol Adlercreutzia, Assacharobacter, Eggerthella, from daidzein can be achieved by all animal species Enterorhabdus, Paraeggerthella, and Slackia tested so far (Schwen et al., 2012). However, only (Clavel et al., 2014). Strains of species such as 25-50% of the people, depending on the human Adlercreutzia equolifaciens, Assacharobacter

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celatus, Enterorhabdus mucosicola, Slackia 2. Material and methods equolifaciens, and Slackia isoflavoniconvertens have 2.1. Ethical approval, donors and faecal sample been reported to be equol producers (Mayo et al., collection 2019). Recent studies using 16S rRNA targeted and shotgun metagenomic techniques have reported a This study was approved by the Bioethics higher relative abundance and prevalence of these Committee of CSIC (Consejo Superior de species in faecal samples from equol producers as Investigaciones Científicas, Spain) and the Regional compared to those from equol non-producers in Ethics Committee for Clinical Research (Servicio de some studies (Iino et al., 2019; Zheng et al., 2019), Salud del Principado de Asturias, Spain) (Ref. but not in others (Nakatsu et al., 2014; Guadamuro 84/14). In compliance with the declaration of et al., 2019). Furthermore, the equol-producing taxa Helsinki, all participants signed a written informed have been shown to be present in the gut microbiota consent. From a group of women enrolled in a of both equol producers and non-producers (Iino et previous work (Vázquez et al., 2017), a total of five, al., 2019; Guadamuro et al., 2019; Zheng et al., categorized on the basis of their equol-producing 2019). This suggests that our knowledge on the phenotype and the presence of equol-related genes, diversity and activity of equol-related taxa in the were selected. Three of the women (W3, W8, and human gut remains poorly understood. W18) produced equol and harboured equol genes. Genomic and proteomic analyses have allowed Another woman (W5) did not produce equol and did deciphering the biochemical and genetic machinery not contain equol-related genes. The last woman involved in equol production in some bacteria. The (W7) proved negative for equol-production but products of three genes (dzr, ddr, and tdr) encoding harboured equol-related genes. Stool samples were reductases, namely daidzein-dependent NADP self-collected by the five volunteers in sterile reductase, dihydrodaidzein reductase, and containers with Anaerocult A (VWR International, tetrahydrodaidzein reductase, respectively, proved to Radnor, PA, USA), transported to the laboratory in be the minimal requirement for equol production less than 2 h, and stored at -80ºC until analysis. (Shimada et al., 2010; Schröder et al., 2013). As it 2.2. Total DNA extraction happens with the equol-producing taxa, the genes involved in equol biosynthesis have also been found For total microbial DNA extraction, 0.2 g of in the faeces of both equol producers and equol non- faeces were suspended in 1.8 mL of phosphate- producers (Braune and Blaut, 2018; Vázquez et al., buffered saline (PBS; VWR International). To 2017). More studies are thus required to disentangle eliminate solid and insoluble wastes and intestinal the lack of correlation between the equol-producer cells, the homogenized suspensions were centrifuged phenotype and the presence of equol-related bacteria at low speed (800 rpm) for 10 min at 4ºC. and genes. Supernatants were transferred to a new tube and In this work, a metagenomic shotgun sequencing centrifuged at 14,000 rpm for 5 min at 4ºC. Then, approach was undertaken to gain insights into the bacterial pellets were washed twice with PBS, and feasibility of this technique for studying the total DNA was extracted and purified following the microbial and genetic characteristics of equol protocol described by Zoetendal et al. (2006) with production. To this purpose, similarities and some modifications as reported by Yu and Morrison differences in the taxonomic composition of the (2004). In short, bacterial pellets were suspended in faecal microbiota of a selected group of five 200 µL of a solution containing 20 mM Tris-HCl pH -1 postmenopausal women with a known equol- 8.0, 2 mM EDTA, 1.20% Triton X-100, 20 mg mL producing phenotype, three equol producers and two lysozyme (Merck, Darmstadt, Germany), 20 U equol non-producers, was undertaken by the use of a mutanolysin (Sigma-Aldrich, Saint Louis, MS, -1 high throughput sequencing strategy. This technique USA), and 10 mg mL of lysostaphin (Sigma- may further allow us to obtain in-depth information Aldrich) and incubated for 40 min at 37ºC. In on the functional pathways encoded by the addition to chemical lysis, cells were disrupted with metagenome of the faecal microbial communities in zirconia-silica beads (0.1 and 0.5 mm) using a equol producers and non-producers, with particular FastPrep FP120 Cell Disrupter (Qbiogene, Carlsbad, -1 attention to genes involved in equol production. CA, USA) at 5.5 m s for 30 s; this procedure was repeated three times with a cooling step of samples

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on ice for 1 min between repetitions. After lysis, the minimum length of 120 bases were considered for DNA was precipitated overnight at -20ºC with 1/4 further analysis) using PRINSEQ-Lite v0.20.4 volumes of 10 M of ammonium acetate (Merck) and (Schmieder and Edwards, 2011). Clean sequences 1 volume of 2-propanol (Sigma-Aldrich). After were screened against the Human reference genome centrifugation at 13,300 rpm for 15 min, the DNA (GRCh37d5 Homo sapiens) downloaded from pellet was suspended in 100 µL of Tris-EDTA (TE) Illumina iGenomes (https://support.illumina. buffer and incubated with 0.2 mg mL-1 RNase and com/sequencing/sequencing_software/igenome.html then with 3 mg mL-1 proteinase K (both from Sigma- , 2019) to remove host reads using BMTagger Aldrich) for 15 min at 37 or 70 ºC, respectively. The (Version 3.101) (Rotmistrovsky and Agarwala, DNA was purified using the QIamp DNA Stool 2011). Paired-end reads were joined and filtered for Minikit (Qiagen, Hilden, Germany) and eluted with duplicates using Picard tools 2.7.1, specifically 150 µL of sterile molecular-grade water (Sigma- FastqToSam and MarkDuplicates tools, respectively Aldrich). The concentration and quality of the DNA (“Picard Toolkit” 2019, Broad Institute, GitHub was determined in an Epoch microvolume Repository, http://broadinstitute.github.io/picard/). spectrophotometer (BioTek Instruments, Winooski, Reads were then trimmed for low quality score using VT, USA) to comply with the parameters required a modified version of the script trimBWAstyle.pl for shotgun sequencing (DNA concentration >300 that works directly from BAM files ng/µL, ratios of A260/280 >1.5 and A26/230 >1.5). (TrimBWAstyle.usingBam.pl, 2010; https://github. Finally, DNA samples were stored at -20ºC until com/genome/genome/blob/master/lib/perl/Genome/ required. Site/TGI/Hmp/HmpSraProcess/trimBWAstyle.using Bam.pl). The script was used to trim off bases with a 2.3. Library construction and shotgun sequencing quality value of three or lower. This threshold was Library construction and sequencing were chosen to delete all the bases with an uncertain carried out at Life Sequencing (Valencia, Spain). quality as defined by Illumina’s EAMMS (End Briefly, 150 pg of total DNA from each faecal Anchored Max Scoring Segments) filter. The sample was employed to prepare shotgun analysis of the microbial composition with the clean metagenomic sequencing using the Nextera XT reads was carried out using MetaPhlAn2 (Segata et DNA Library prep kit (Illumina, San Diego, CA, al., 2012) species classifier against default USA). Fragmented DNA was purified using the parameters on the mpa_v20_m200 marker database, AMpure XP System (SPRI beads) (Beckman and Kraken 2 (Wood and Salzberg, 2014) using a k- Coulter, Brea, CA, USA), and validated and mer matching algorithm run against the Standard quantified using a Qubit spectrofluorometer Kraken 2 Database containing all RefSeq for the (Thermo Fisher Scientific, Waltham, MA, USA) and bacterial, archaeal, and viral domains. According to an Agilent 2100 Bioanalyzer (Agilent Technologies, Lu et al. (2017), Bracken, using Kraken 2 outputs, Santa Clara, CA, USA). After passing quality was applied to re-estimate bacterial abundances. checks, libraries were prepared with Nextera XT Taxa with a relative abundance of <0.1% were DNA library prep kits (Illumina), and the samples categorized as “Other” for each classifier. were sequenced on the Illumina HiSeq™ 500 Subsequently, function of the reads was assigned platform (2 x 150-bp reads), following standard with HUMAnN2 tool based on ChocoPhlan and Illumina sequencing protocols. UniRef databases. The HUMAnN2 gene abundance

2.4. Sequences and data analysis table was regrouped by a mapping of GO terms for all categories of bacterial metabolism and dividing Sequencing data were processed with the the functional table into two files (one stratified and bcl2fastq 2.20 conversion software one non-stratified). The SUPER-FOCUS (Silva, (https://support.illumina.com/downloads/bcl2fastq- G.G.Z. et al., 2016) program with the help of the conversion-software-html) to eliminate adapters and RAPSearch and DIAMOND programs to reads having a Phred quality (Q) score lower than functionally assign the clean reads was also used. 20. The resulting fastq files were preprocessed by Filtered reads were assembled using IDBA_UD sequence quality (removal all bases at the 3’ end (kmers 20-120) (Peng et al., 2012), keeping those with an average Phred score < 25 over a sliding contigs with length above 500 bp. To look for the window of 10 bp) and length (only reads with a

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presence of equol-related genes in the samples, the significant differences in microbiota composition NCBI BLAST tool was used, taking as a reference were assessed by PERmutational Multivariate all the sequences deposited in the NCBI database ANalysis Of VAriance (PERMANOVA) from (consulted in June 2020) of the three reductase genes Vegan (Adonis 2 function) (Oksanen et al,. 2018) (dzr, ddr and tdr). In the analysis of the sequences using a Bray-Curtis dissimilarity measure. using the NCBI BLAST tool, high homology filters Continuous variables were described as mean and were used. standard deviation (SD) or median. Statistical significance was established at P<0.05. 2.5. Data statistical analysis 2.6. Submit sequence data to NCBI All statistical analyses were performed with R version 3.6.0 (https://www.r-project.org/). Normality The raw data obtained in this study were of the data was evaluated with Shapiro-Wilk test, deposited in the Sequence Read Archive (SRA) of and ANOVA was used to calculate the significance the NCBI database (http://www.ncbi.nlm.nih.gov) in the analysis of alpha-diversity index. Statistically under Accession Number…

Figure 1.- Microbial composition of faecal samples from women of equol-producing and equol non-producing phenotype using two different classifiers: MetaPhlAn2 (A) and Bracken (B). Each stacked bar plot shows the relative abundances of bacterial community at species level (>0.1%).

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3. Results from volunteers of the two phenotypes were not observed (P=0.4 for Shannon index and P=0.8 for 3.1. Sequencing data Simpson index) (Supplementary Figure 1). After trimming of low quality reads and removal Additionally, to determine whether there was of human-derived sequence contaminants (which dissimilarity between samples in microbial relative ranged from 0.16 to 1.15% of the reads per sample), abundances at the family, genus and species levels, a a mean of 2 , 39,0 ± 2,432,955 reads per sample non-metric multidimensional scaling (NMDS) were obtained with a length ranging from 146 to 150 analysis was conducted (Supplementary Figure 2). bp and a Phred quality (Q) average of 32 When using data from MetaPhlAn2, the NMDS (Supplementary Table 1). After assembly, data analysis revealed that the bacterial community yielded a mean of 79,728 ± 29,091 contigs per structure of faeces from the equol-producing and sample with a length greater than 500 bp. equol non-producing women segregated at all levels (Supplementary Figure 2, A1-A3). However, using 3.2. Taxonomic bacterial composition data from Bracken, the sample coming from the To search for differences in community equol non-producing woman harbouring equol- composition of the microbiota between equol related genes clustered within the samples from producers and equol non-producers, alpha diversity equol-producing women (Supplementary Figure 2, indices were calculated. Significant differences in B1-B3). overall bacterial richness between faecal samples

A B

W3 W7 W8 W5 W18 W7 W8 W18 W5 W3 Figure 2.- Heat map diagram correlating relative abundances of bacterial species identified by MetaPlan2 (A) or Bracken (B) in the faecal microbiota of equol-producing and non-producing women. Asterisk indicates presence of a certain species exclusively or in large abundance in equol producer women.

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Important differences in taxonomic assignation present or in large abundance in samples from were found between the results obtained with women with an equol-producing phenotype: MetaPhlAn2 and Bracken (Figure 1 and Alistipes putredinis, Bacteriodales bacterium Supplementary Figure 3). In general, taxonomic (renamed as Alistipes communis), and Bilophila sp. analysis (combining results obtained by both (Figure 2A). On the contrary, species present only in classifiers) revealed that most sequences belong to samples from equol-producer women were not the species Eubacterium rectalis (recently assigned detected by Bracken analysis (Figure 2B). to Agathobacter rectalis), Faecalibacterium 3.3 Equol-producing related species prausnitzii, Roseburia hominis, Parabacteroides distasonis, and Bifidobacterium longum (Figure 1; Most equol-producing bacteria identified so far Supplementary Figure 3). Heat map analysis using belongs to the class Coriobacteriia and the family species with > 0.1% abundance within samples did Eggerthellaceae of the phylum Actinobacteria. not show any significant correlation between Reads of the Coriobacteriia class were detected in microbial populations and equol production status samples from both equol-producing and equol non- (Figure 2). However, when conducting manually a producing women; their relative abundance ranging more detailed Heat map evaluation, MetaPhlAn2 from 0.9 to 3.3 % of the total reads (Figure 3A). analysis identified the presence of three species only

Figure 3.- Relative abundances of Coriobacteriia and equol-producing bacteria in faecal samples from equol producer and equol non-producer women (A). Distribution of species belonging to the class Coriobacteriia in the faecal microbiota of equol producing and non-producing women (B).

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The highest number of reads belonging to the class functional diversity was slightly higher in samples Coriobacteriia was detected in samples from two from equol-producing women as compared to those equol-producing women, W3 and W18. However, from equol non-producers, although differences the lowest percentage (0.9%) was also quantified in were not statistically significant (P=0.4 for Shannon the faeces of an equol producer (W8). At the species index and a P=0.78 for Simpson index; level, A. equolifaciens, Eggerthella lenta, and Supplementary Figure 4A). Carbohydrate Gordonibacter pamelaeae were the dominant metabolism, protein and amino acids metabolism, Coriobacteriia species detected in all samples nucleotide metabolism, metabolism of cofactors and (Figure 3B). Neither of these nor others species in vitamins, cell wall integrity, membrane transport, lower abundance were exclusively associated with stress response and virulence were found as the most the equol-producing phenotype. A. equolifaciens and abundant functional categories in the microbiota of Eggerthella sp. YY918 species, for which some both producing and non-producing women; these strains have been reported to be equol producers, modules constituted more than 62,13% of total were also identified in all samples. Indeed, the metagenomics reads (Supplementary Figure 4B). Of relative abundance of these equol-producing bacteria the 31 super-pathways or pathways showing at least did not correlate with the equol-producing 1-fold difference in abundance between phenotypes, phenotype. 18 pathways were enriched in the faecal metagenome of equol producers, while 13 were 3.4. Functionality of the microbiota enriched in the metagenome of equol non-producers Considering the annotations with HUMAnN2 (Figure 4). The largest increase in the metagenome and SUPER-FOCUS programs, the number of of equol producers, listed in order of abundance, was different metabolic pathways (Subsystem level 2), for enzymes involved in tricarboxylic acid (TCA) with a relative abundance larger than 0.001%, cycle, L-glutamate degradation, and glyoxylate ranged in the samples from 139 to 153. Overall, the cycle.

Figure 4.- Difference in relative abundances (> 1-fold unit) of metabolic pathways in the gut microbiota of equol-producing and equol-non-producing women. Colours code: pathways over-represented (in blue) or under-represented (in orange) in equol-producing women.

59 3.5. Equol-related genes due to the K-mer-based methodology (Rajan et al., 2019), which further enlarges the possibility of As HUMAnN2 and SUPER-FOCUS programs misclassifying close-related taxa. Therefore, the did not identify sequences encoding equol choice of combining different methodologies can be production-related pathways or enzymes, attempts a good option to reach robust results in sample were made by manual Blast search using as comparison until the development of more accurate references all sequences of genes encoding bioinformatics pipelines for analysis of shotgun reductases (dzr, ddr and tdr) involved in equol sequencing data sets. In this sense, Lugli and co- production and deposited at the NCBI database workers have recently pointed out that the results (accessed in June 2020). Among the metagenomics obtained by next-generation sequencing (NGS) data, only a single sequence highly homologous to a should be subsequently confirmed by culturing segment of the dzr gene from A. equolifaciens (Lugli et al., 2019). DSM19450T was found in the sample from the Daidzein intake by equol producers have already equol-producing woman W14. When more been correlated with significant increases in permissive Blast parameters were selected, reads of sequences of taxa such as Eubacterium, FAD-dependent oxidoreductase- and glutamate Faecalibacterium, Clostridium, and Collinsella synthase-encoding genes showing low homology to (Nakatsu et al., 2014; Guadamuro et al, 2015). In the reductases involved in equol biosynthesis were this study, these genera were also found to be more identified. However, these were present at the same abundant in samples from equol producers. numbers in samples from both equol producers and However, high numbers of sequences of these equol non-producers. Finally, metagenome data genera were also detected in samples from equol were matched against sequences of segments of the non-producing women. At the species level, tdr gene obtained previously by standard PCR from significant differences in the samples of equol the faeces of each equol-producing women producers and non-producers were only found in (Vázquez et al., 2017), and identical results were subdominant populations. As a result, a clear profile obtained. based on a core of bacterial species associated with the equol-producing phenotype was not observed. 4. Discussion Nonetheless, reads of A. putredinis, B. bacterium Equol production from daidzein is due to the (renamed A. communis), and Bilophila sp. species metabolism carried out by certain bacteria in the gut. were found exclusively or in a large abundance in However, the gut microbiota composition and the samples from all equol producers. These species metabolic pathways involved in equol production have been previously correlated with a healthy gut are poorly understood. To delve into these issues, in microbiome (Le Chatelier et al., 2013; Kuhn et al., this study, the relationships among compositional 2018; Averina et al., 2020; Wu et al., 2020). and functional diversity of gut microbiota and equol- However, their potential role on the metabolism of producing phenotype were assessed by a shotgun isoflavones or their relationship with equol- sequencing approach. After shotgun sequencing of producing bacteria remains to be determined. E. total microbial DNA from faecal samples, two rectale, a species that has been previously associated standard phylogenetic classification methods, with isoflavones consumption and the underlying MetaPhlAn2 and Bracken, were used to investigate equol excretion levels (Clavel et al., 2005), was the community structure of the gut microbiota from detected as the majority population in samples from equol-producing and equol non-producing women. two equol-producing women (W3 and W8), but also The comparative of both methods revealed large in that from one equol non-producer (W7). differences in bacterial taxonomic classification, Sequences belonging to A. equolifaciens and especially, at genus and species levels; this likely Eggerthella sp., for which some strains have been lies in the distinct assignment strategy described as equol-producing microbes (Maruo et followed by MetaPhlAn2 and Bracken. While al., 2008; Yokoyama and Suzuki, 2008), were MetaPhlAn2 only profiles taxa included in the clade- present in faecal samples of both equol producers specific marker gene database (Lugli et al., 2019), and equol non-producers. This agrees with previous which tends to under-estimate some bacterial studies in which equol-related taxa had been populations, Bracken seems to over-represent them

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detected in samples from both equol producers and inoculated with faeces from equol producers non-producers (Nakatsu et al., 2014; Sugiyama et (Guadamuro et al., 2017). In addition, the al., 2014; Guadamuro et al, 2015; Iino et al., 2019; conversion of daidzein to equol by equol-producing Zheng et al., 2019). In this study, the relative strains has been demonstrated to be boosted in vitro abundance of equol-producing bacteria showed large by the addition of butyric and propionic acids inter-individual differences across samples from (Decroos et al., 2005; Minamida et al., 2006). Small different women. However, a higher amount of increases of sequences with homology to genes sequences did not correlate with an equol-producing coding for enzymes involved in the synthesis of status. Recently, sequencing and analysis of A. isopentenyl diphosphate and geranylgeranyl equolifaciens IPLA37004 genome revealed a diphosphate (precursors of vitamin A; Srinivasan deletion of the whole equol operon region (as and Buys, 2019), as well as lipopolysaccharides and compared to the equol-producing strain A. enzymes that take part in the 2-methylcitrate cycle equolifaciens DSM19450T) (Vázquez et al., 2020). and methanol oxidation reactions (Upton and Thus, the presence in the microbiota of species McKinney, 2007), have also been detected in the considered, to date, as equol-producing taxa may not metagenome of equol-producing women. Nothing is to be directly associated with equol production. This known about the connection that exits between a result suggests that the equol-producing phenotype higher abundance of these pathways and the equol cannot be entirely based on taxonomic analysis. phenotype. The data obtained by shotgun sequencing were Surprisingly, the functional metabolic analysis analysed by HUMAnN2 and SUPER-FOCUS failed to detect sequences related to equol software programs to identify enriched metabolic production. Miss-annotation of reductases or other pathways in equol-producing women and evaluate equol-related genes in databases could impair their links with equol production. The metabolic accurate detection of these genes. To avoid this pathways showed slight differences between trouble, all sequences of genes deposited in samples from producers and non-producers, which databases encoding the key reductases involved in correlated with the scarce variations in the bacterial equol synthesis (dzr, ddr, and tdr) were used to populations observed in the taxonomic analysis. The search for equivalent sequences in the metagenomics gut microbiota of equol-producing women showed, data. Only a single sequence with high homology to as compared to non-producers, a moderate increase the daidzein reductase gene (dzr) from A. of enzymes involved in tricarboxylic acid (TCA) equolifaciens DSM19450T was found in the cycle, as well, in L-glutamate degradation, and metagenome of an equol-producing woman. The glyoxylate cycle. The TCA cycle plays a central role difficulty for detecting such sequences might be in the catabolism of carbohydrates, fats and proteins, based on the low number of genes involved in equol and provides certain amino acids and the reducing production within the whole faecal metagenome agent NADH required in multiple cellular reactions. (104-105 copies/gr of faeces; Vázquez et al., 2017). A recent metagenome analysis has shown that green In that sense, the presence of high amounts of host tea polyphenols administration increased a number DNA in faecal samples has been demonstrated to of genes of the gut microbiota associated with the decrease sequencing depth and, therefore, led to a TCA cycle and ATP synthesis (Zhou et al., 2020). In reduction in detecting low or very low abundant contrast to our observations, the increases correlated sequences (Pereira-Marques et al., 2019). In with higher abundance of bacteria from the families accordance with this, sequences encoding FAD- Ruminococcaceae, Lachnospiraceae, and dependent oxidoreductases and glutamate synthases Bacteroidaceae. TCA, L-glutamate and glyoxylate sharing certain homology to equol-related genes, but pathways may contribute to butyrate and propionate widely spread among many bacterial taxa (and formation from carbohydrates in the lumen of the therefore present in higher numbers), were colon (Louis and Flint, 2017; Ahn et al., 2016). The abundantly detected. increase of sequences related to these cycles in women with an equol-producing phenotype agrees Conclusions with the higher butyric and In summary, the gut bacterial communities of production that has been observed in faecal cultures equol producers and equol non-producers tested in

61 this study were practically identical, even as regards 2007. Microbial and dietary factors are associated with to taxa related to equol production. Species of the the equol producer phenotype in healthy Alistipes and Bilophila genera were the only ones postmenopausal women. Journal of Nutrition, 137, detected to be present exclusively or in higher 2242–2246. abundance in the faeces of equol-producing women. Braune, A., and Blaut, M. 2018. Evaluation of inter- However, the relationship between these species and individual differences in gut bacterial isoflavone bioactivation in humans by PCR-based targeting of the equol-producing phenotype is uncertain. genes involved in equol formation. Journal of Applied Functional analysis of the metagenomic data failed Microbiology, 124, 220–231. to detect pathways involved in isoflavone Clavel, T., Fallani, M., Lepage, P., Levenez, F., Mathey, metabolism and equol production. A specific search J., Rochet, V., Sérézat, M., Sutren, M., Henderson, G., for reductase-encoding genes involved in equol Bennetau-Pelissero, C., Tondu, F., Blaut, M., Doré, J., production revealed the presence in the metagenome and Coxam, V. 2005. Isoflavones and functional foods of one equol producer of a single read highly alter the dominant intestinal microbiota in homologous to a segment of a daidzein reductase postmenopausal women. The Journal of Nutrition, gene (dzr). Further development of shotgun 135(12), 2786–2792. techniques and/or computational approaches are still Clavel, T., Lepage, P., and Charrier, C. 2014. The family required to overcome the challenges that affect the Coriobacteriaceae. In, The Prokaryotes- detection of genes involved in equol production in Actinobacteria. Rosenberg, E., DeLong, E.F., Lory, S., faecal samples. Stackebrandt, E., Thompson, F., Eds.; Springer: Berlin/Heidelberg, Germany. pp. 201–238. Acknowledgments Decroos, K., Vanhemmens, S., Cattoir, S., Boon, N., and Verstraete, W. 2005. Isolation and characterisation of This study was funded by projects from the an equol-producing mixed microbial culture from a Spanish Ministry of Economy and Competitiveness human faecal sample and its activity under (MINECO) (AGL2014-57820-R) and the gastrointestinal conditions. Archives of Microbiology, Principality of Asturias (IDI/2018/000114). LV was 183(1), 45–55. supported by a research contract from the FPI Franke, A.A., Lai, J.F., and Halm, B.M. 2014. Absortion, Program and from MINECO (BES-2015-072285). distribution, metabolism, and excretion of isoflavonoids after soy intake. Archives of References Biochemistry and Biophysics, 59, 24–28. Guadamuro, L., Delgado, S., Redruello, B., Flórez, A.B., Ahn, S., Jung, J., Jang, I. A., Madsen, E. L., and Park, W. Suárez, A., Martínez-Camblor, P., and Mayo, B. 2015. 2016. Role of Glyoxylate shunt in oxidative stress Equol status and changes in fecal microbiota in response. The Journal of Biological Chemistry, menopausal women receiving long-term treatment for 291(22), 11928–11938. menopause symptoms with a soy-isoflavone Averina, O.V., Kovtun, A.S., Polyakova, S.I., Savilova, concentrate. Frontiers in Microbiology, 6, 777. A.M., Rebrikov, D.V., and Danilenko, V.N. 2020. The Guadamuro, L., Dohrmann, A.B., Tebbe, C.C., Mayo, B., bacterial neurometabolic signature of the gut and Delgado, S. 2017. Bacterial communities and microbiota of young children with autism spectrum metabolic activity of faecal cultures from equol disorders. Journal of Medical Microbiology, 69(4), producer and non-producer menopausal women under 558-571. treatment with soy isoflavones. BMC Microbiology, Bilal, I., Chowdhury, A., Davidson, J., and Whitehead, S. 17(1), 93. 2014. Phytoestrogens and prevention of breast cancer: Guadamuro, L., Azcárate-Peril, M. A., Tojo, R., Mayo, B., The contentious debate. World Journal of Clinical & Delgado, S. (2019). Changes in the faecal Oncology, 5, 705–712. microbiota of an equol-producing menopausal woman Bolaños, R., Del Castillo, A., and Francia, J. 2010. Soy over six months of dietary supplementation with isoflavones versus placebo in the treatment of isoflavone. AIMS Microbiology, 5, 102–116. climacteric vasomotor symptoms: Systematic review Hall, M.C., O’Brien, B., and McCormack, T. 200 . Equol and meta-analysis. Menopause, 17, 660–666. producer status, salivary estradiol profile and urinary Bolca, S., Possemiers, S., Herregat, A., Huybrechts, I., excretion of isoflavones in Irish Caucasian women, Heyerick, A., DeVriese, S., Verbruggen, M., following ingestion of soymilk. Steroids, 72, 64–70. Depypere, H., De Keukeleire, D., Bracke, M., De Iino, C., Shimoyama, T., Iino, K., Yokoyama, Y., Chinda, Henauw, S., Verstraete, W., and Van de Wiele, T. D., Sakuraba, H., Fukuda, S., and Nakaji, S. 2019.

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MATERIAL SUPLEMENTARIO

A

B

Supplementary Figure 1: Box-plots representing alpha diversity indices (Shannon diversity, Simpson index and Richenest) of identified species, using MethaPhlAn2 (A) and Bracken (B), in the faecal microbiota of women with an equol and equol non-producing phenotype.

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A1 A2 A3

B1 B2 B3

Supplementary Figure 2.- Non-metric Multidimensional Scaling (NMDS) analysis between the equol producers (green circles) and non-producers (orange circles) using taxonomic data from MethaPhlAn2 (A) and Bracken (B) analysis at family (A1, B1), genus (A2, B2), and species (A3, B3) levels.

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A1 A2

B1 B2

Supplementary Figure 3.- Microbial composition of faecal samples from women of equol-producing and equol non-producing phenotype using two different classifiers: MetaPhlAn2 (A) and Bracken (B). Each stacked bar plot shows the relative abundances (>1%) of bacteria at phylum (A1; B1), and genus (A2; B2) levels.

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A

B

Supplementary Figure 4.- Alpha diversity indices (Shannon diversity, Simpson index and Richenest) in the metagenome of faecal samples from women of equol-producing and equol non- producing phenotype (A). Distribution and percentage of sequences assigned to different metabolic functions in samples from equol producer and equol non-producer women (B).

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Supplementary Table 1.- Summary of metagenomics data obtained after shotgun sequencing, removal of human-derived contaminant sequences, and assembly of reads from the faecal samples of equol producer and equol non-producer women of this study.

Average Nº of total Nº of contigs Sample read lenght Q media reads (>500pb) (bp)

Equol producing women W3 27.305.010 146.7 32.64 90,849

W8 22,621,676 147.5 32.83 57,082

W18 28,670,800 149.6 32.21 109,390

Equol non-producing women W5 27,318,278 150,3 32.80 99,852

W7 28,286,592 147.8 32.58 41,467

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CAPÍTULO 2

Estudio de las relaciones e interacciones entre isoflavonas, equol y poblaciones bacterianas intestinales.

En este capítulo se recogen los resultados de tres trabajos en los que se estudia el efecto de las isoflavonas de la soja sobre las poblaciones bacterianas mayoritarias del TGI, se profundiza en el metabolismo de las isoflavonas por parte de microorganismos intestinales de taxones dominantes y subdominantes del TGI y, por último, se describe la secuenciación genómica de la cepa aislada en este trabajo, Adlercreutzia equolifaciens IPLA37004.

. Artículo III.- Vázquez, L., Flórez, A. B., Guadamuro, L. y Mayo, B. (2017). Effect of soy isoflavones on growth of representative bacterial species from the human gut. Nutrients, 9(7), 727. doi:10.3390/nu9070727.

. Artículo IV.- Vázquez, L., Flórez, A. B., Redruello, B. y Mayo, B. (2020). Metabolism of soy isoflavones by intestinal bacteria: genome analysis of an Adlercreutzia equolifaciens strain that does not produce equol. Biomolecules, 10(6), 950. doi: 10.1016/j.jff.2020.103819.

. Artículo V.- Vázquez, L., Flórez, A. B. y Mayo, B. (2020). Draft genome sequence of Adlercreutzia equolifaciens IPLA 37004, a human intestinal strain that does not produce equol from daidzein. Microbiology Resource Announcements, 9(8), e01537-19. doi:10.1128/MRA.01537-19.

En el primer artículo se encontró que la mayoría de las formas glicosiladas de las isoflavonas (daidzina y genistina), las agliconas (daidzeína y genisteína) y el equol apenas presentan actividad antimicrobiana a concentraciones superiores a las fisiológicas frente a bacterias ácido-lácticas, bifidobacterias y otras especies dominantes presentes en el tracto gastrointestinal. No obstante, los resultados obtenidos reflejan que las isoflavonas y su metabolitos derivados podrían tener la capacidad de modular las poblaciones, mediante una reducción especie- o cepa específica de la tasa de crecimiento.

El análisis de la actividad metabólica de diversos biotipos aislados de heces de mujeres productoras de equol demostró que algunos de ellos son capaces de transformar las isoflavonas generando pequeñas cantidades de dihidrodaidzeína y O-desmetilangolesina a partir de la daidzeína, y dihidrogenisteína a partir de genisteína. Sin embargo, a pesar de

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tener que estar presentes, ninguno de los aislados obtenidos produjo equol, y eso a pesar de que una de las cepas (IPLA 37004) se identificó como Adlercreutzia equolifaciens que, como refiere su nombre, está descrita como productora de equol. El análisis y la comparación de las secuencias del genoma de A. equolifaciens IPLA37004 y A.equolifaciens DSM19450T (bacteria productora de equol) reveló en la primera la ausencia del clúster de biosíntesis de equol. El análisis de los genomas de distintas cepas pertenecientes a las especies productoras de equol A. equolifaciens y Asaccharobacter celatus sugirió que en el TGI de mujeres productoras de equol podrían convivir representantes de los mismos taxones productores y no productores de equol.

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Article Effect of Soy Isoflavones on Growth of Representative Bacterial Species from the Human Gut

Lucía Vázquez, Ana Belén Flórez, Lucía Guadamuro and Baltasar Mayo *

Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain; [email protected] (L.V.); abfl[email protected] (A.B.F.); [email protected] (L.G.) * Correspondence: [email protected]; Tel.: +34-985-89-21-31

Received: 3 May 2017; Accepted: 4 July 2017; Published: 8 July 2017

Abstract: The present work aimed to assess the susceptibility of dominant and representative bacterial populations from the human gut to isoflavones and their metabolites. To do so, the minimum inhibitory concentration (MIC) of isoflavone glycosides, isoflavone aglycones, and equol to 37 bacterial strains was determined by broth microdilution. Additionally, for 10 representative strains, growth curves, growth rate (µ), and optical density (OD600 nm) of the cultures at 24 h were also determined. MICs of daidzin, genistin, daidzein, and genistein were >2048 µg mL−1 for all strains assayed, while that of equol ranged from 16 µg mL−1 for Bifidobacterium animalis subsp. animalis to >2048 µg mL−1 for Enterobacteriaceae strains. Changes in growth curves, µ, and final OD were observed among the species in the presence of all tested compounds. Genistein reduced µ of Bacteroides fragilis, Lactococcus lactis subsp. lactis, and Slackia equolifaciens, while both genistein and equol increased that of Lactobacillus rhamnosus and Faecalibacterium prausnitzii. Compared to controls, lower final OD in the presence of aglycones and equol were recorded for some strains but were higher for others. Altogether, the results suggest that isoflavone-derived compounds could modify numbers of key bacterial species in the gut, which might be associated with their beneficial properties.

Keywords: isoflavones; daidzein; genistein; equol; minimum inhibitory concentration; lactic acid bacteria; bifidobacteria; intestinal bacteria

1. Introduction High intakes of soy-containing foods have been epidemiologically associated with less intense menopausal symptoms and a reduced risk of developing cardiovascular and neurodegenerative diseases and cancer [1]. Though soy contains many biologically active substances [2], its beneficial health effects have been attributed to its isoflavone content [3]. Isoflavones are phenolic compounds found naturally in plants (among which soy is one of the richest sources), the chemical structures of which resemble 17-β-oestradiol. They therefore have estrogenic effects [3]. In soy milk and unfermented soy foods, isoflavones mostly appear as isoflavone–glycoside conjugates (daidzin, genistin, glycitin)—the bioavailability and estrogenic activity of which are low [4]. To be absorbed and reach full activity, isoflavone aglycones (daidzein, genistein, glycitein) need to be released from the corresponding glycosides [5]. This is accomplished by cellular β-glucosidases and β-glucosidases from components of the gut microbiota [6]. Isoflavone aglycones can be metabolized further by cellular enzymes, plus others from components of the gut microbiota [7], to produce more active compounds (such as equol from daidzein) or inactive metabolites [8]. Some of the beneficial health effects attributed to isoflavones could come about via the stimulatory or inhibitory modulation of gut microbial populations. However, the effects of isoflavones on gut microbiota have been little examined [9–12]. Increases in the number of bifidobacteria have been

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Nutrients 2017, 9, 727 recorded in some studies [10,12], and population sizes within Clostridium clusters have been reported to increase in equol producers [10,13]. However, reductions in bifidobacteria and populations of Enterobacteriaceae have been observed in other studies [11]. Such contradictory results may ultimately depend on the baseline size and composition of the bacterial communities in the gut, which can vary widely between subjects [14]. Finally, like many other polyphenols [15], isoflavones and some of their metabolites have been shown to possess a certain antimicrobial activity against bacterial pathogens [16–18]. It is thus conceivable that they might directly or indirectly alter the numbers or relative proportions of pivotal bacterial communities for maintaining a healthy microbial balance in the gut. The present work aimed to examine the possible inhibitory effect of the most common soy isoflavone glycosides (daidzin and genistin), their derived aglycones (daidzein and genistein), and equol, against 37 bacterial strains, including lactic acid bacteria, bifidobacteria, and strains of other dominant and representative bacterial groups in the human gut.

2. Materials and Methods

2.1. Bacterial Strains, Growth Media, and Culture Conditions Of the 37 strains used in this study, 25 were type strains of lactic acid bacteria (LAB) and bifidobacterial species obtained from the Laboratory of Microbiology collection in the Belgian Coordinated Collections of Microorganisms (BCCM/LMG) (Ghent University, Ghent, Belgium), 7 were strains (of which 6 were type strains) of species from human intestines obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) (Leibniz Institute, Braunschweig, Germany), and 5 strains of intestinal species were from our own laboratory collection (Table 1). Strains were considered representative of functional bacterial groups within the human gut; they also represent those most commonly used as probiotics. Lactococci were grown on M17 agar (Oxoid, Basingstoke, UK) supplemented with 1% glucose (VWR International, Radnor, PA, USA) at 32 ◦C for 48 h under aerobic conditions. Streptococcus thermophilus was cultured on M17 agar (Oxoid) supplemented with 1% lactose (VWR International) at 37 ◦C for 48 h, under anaerobic conditions. Heterofermentative lactobacilli were recovered on de Man, Rogosa, and Sharpe (MRS) (Merck, Darmstad, Germany) agar plates and incubated for 48 h at 32 ◦C or 37 ◦C and under aerobic or anaerobic conditions, depending on the species. Homofermentative lactobacilli and bifidobacteria ◦ were grown on MRS agar supplemented with 0.25% L-cysteine (Merck) and incubated at 37 C for 48 h under anaerobic conditions. Intestinal anaerobic strains (Bacteroides spp., Blautia coccoides Faecalibacterium prausnitzii, Ruminococcus obeum, and Slackia spp.) were streaked on Gifu anaerobic medium (GAM) (Nissui, Tokyo, Japan). All strains of these species were incubated at 37 ◦C for 48 h under anaerobic conditions. Finally, strains of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Serratia marcescens were grown on brain heart infusion (BHI; Oxoid) agar at 37 ◦C for 24 h under aerobic conditions.

2.2. Determination of Minimum Inhibitory Concentration The minimum inhibitory concentration (MIC) of the majority of isoflavone glycosides in soy (daidzin and genistin), their respective aglycones (daidzein and genistein), and the isoflavone metabolite equol (all from LC Laboratories, Woburn, MA, USA) were determined using a broth microdilution test, following standard procedures for aerobic [19] and anaerobic bacteria [20] with minor modifications. Briefly, individual colonies from the above plates were suspended in 5 mL of a sterile 0.9% NaCl solution (VWR International) to a McFarland turbidity of 1. The inoculated saline solution was then diluted 1:1000 in the test medium corresponding to the different species (see Table 1) to obtain an approximate final concentration of 3 × 105 cfu mL−1. Aliquots (100 µL) of the diluted cell suspensions were poured into microplate wells with 50 µL of two-fold increasing concentrations of the test compounds, ranging from 0.12 to 2048 µg mL−1 (the limit of their solubility). MICs were

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Nutrients 2017, 9, 727 established by visual inspection as the lowest concentration at which no visible growth was observed. All MIC assays were performed in duplicate. Where discrepancies between analyses were observed, a third assay was performed and the mode reported.

Table 1. Bacterial strains, assay conditions for the minimum inhibitory concentration (MIC), and MIC results of equol to the intestinal species and strains under study.

Bacterial Strains MIC Assay Conditions MIC Results Bacterial Group/Species Strain Code Medium Temperature Atmosphere Equol (µg mL−1) Lactic acid bacteria Lactococcus (Lc.) lactis subsp. cremoris LMG 6987T IST a 32 ◦C Aerobiosis 256 Lc. lactis subsp. lactis LMG 6890T IST 32 ◦C Aerobiosis 128 Streptococcus termophilus LMG 6896T IST-Lac b 37 ◦C Anaerobiosis 256 Lactobacillus (Lb.) brevis LMG 6906T LSM c 32 ◦C Aerobiosis 256 Lb. casei LMG 6904T LSM 32 ◦C Aerobiosis 1024 Lb. fermentum LMG 6902T LSM 37 ◦C Aerobiosis 1024 Lb. paracasei subsp. paracasei LMG 13087T LSM 32 ◦C Aerobiosis 1024 Lb. pentosus LMG 10755T LSM 32 ◦C Aerobiosis 1024 Lb. plantarum LMG 6907T LSM 32 ◦C Aerobiosis 1024 Lb. reuteri LMG 9213T LSM 37 ◦C Aerobiosis 512 Lb. rhamnosus LMG 6400T LSM 37 ◦C Aerobiosis 512 Lb. sakei subsp. sakei LMG 9468T LSM 32 ◦C Aerobiosis 256 Lb. acidophilus LMG 9433T LSM-Cys d 37 ◦C Anaerobiosis 512 Lb. delbrueckii subsp. bulgaricus LMG 6901T LSM-Cys 37 ◦C Anaerobiosis 64 Lb. delbrueckii subsp. delbrueckii LMG 6412T LSM-Cys 37 ◦C Anaerobiosis 256 Lb. delbrueckii subsp. lactis LMG 7942T LSM-Cys 37 ◦C Anaerobiosis 128 Lb. gasseri LMG 9203T LSM-Cys 37 ◦C Anaerobiosis 128 Lb. helveticus LMG 6413T LSM-Cys 37 ◦C Anaerobiosis 1024 Lb. johnsonii LMG 9436T LSM-Cys 37 ◦C Anaerobiosis 512 Bifidobacteria Bifidobacterium (B.) adolescentis LMG10502T LSM-Cys 37 ◦C Anaerobiosis 256 B. animalis subsp. animalis LMG 10508T LSM-Cys 37 ◦C Anaerobiosis 16 B. animalis subsp. lactis E43 LSM-Cys 37 ◦C Anaerobiosis 128 B. breve LMG 13208T LSM-Cys 37 ◦C Anaerobiosis 256 B. longum subsp. longum LMG 13197T LSM-Cys 37 ◦C Anaerobiosis 256 B. pseudolongum subsp. pseudolongum LMG 11571T LSM-Cys 37 ◦C Anaerobiosis 128 B. termophilum LMG 21813T LSM-Cys 37 ◦C Anaerobiosis 256 Other intestinal bacteria Bacteroides (Bact.) fragilis DSM 2151T M1 e 37 ◦C Anaerobiosis 64 Bact. thetaiotaomicron DSM 2079T M1 37 ◦C Anaerobiosis 64 Blautia coccoides DSM 935T M1 37 ◦C Anaerobiosis 256 Faecalibacterium prausnitzii DSM 17677 M1 37 ◦C Anaerobiosis 256 Ruminococcus obeum DSM 25238T M1 37 ◦C Anaerobiosis 256 Slackia (Sl.) equolifaciens DSM 24851T M1 37 ◦C Anaerobiosis 64 Sl. isoflavoniconvertens DSM 22006T M1 37 ◦C Anaerobiosis 1024 Escherichia coli E-73 IST 37 ◦C Aerobiosis 2048 Klebsiella pneumoniae K-78 IST 37 ◦C Aerobiosis 2048 Pseudomonas aeruginosa PS-25 IST 37 ◦C Aerobiosis 1024 Serratia marcescens S-54 IST 37 ◦C Aerobiosis 512 a IST, IsoSensitest (Oxoid); b IST-Lac (IST + 1% lactose); c LSM, Lactic acid bacterium susceptibility test medium (90% IST + 10% de Man, Rogosa and Sharpe (MRS)); d LSM-Cys (LSM + 0.03% cysteine); e M1 (90% IST + 10% Gifu Anaerobic Medium (GAM) + 0.25% cysteine). MICs were assayed in duplicate or triplicate; when discrepancies were found, the mode was reported.

2.3. Effect of Isoflavone Aglycones and Equol on Bacterial Growth The growth of bacteria in the presence of daidzein, genistein, and equol was monitored spectrophotometrically, measuring the optical density (OD) throughout culturing. Colonies were collected, suspended in 10 mL of an appropriate liquid medium, and incubated for 24 h under species-specific conditions as stated above. These cultures were then used to inoculate appropriate fresh media (at 1%) supplemented in independent tubes with daidzein, genistein, or equol (all at 32 µg mL−1). Cultures to which no phenolic compounds were added were used as controls. Growth was monitored by measuring the OD at 600 nm using the culture medium as a blank. All growth experiments were

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Nutrients 2017, 9, 727 performed in triplicate; mean results are reported. The bacterial growth rate (µ) was calculated using the formula µ = Ln(N2/N1)/t2−t1, where N1 was the OD at time 1 (t1) and N2 was the OD at t2. The interval t1-t2 was selected within the logarithmic growth phase of the different species and strains.

2.4. Statistical Analysis Statistical analysis of the data was performed using the 3.2.5. version of the free R software (The R Foundation, Boston, MA, USA). Normality of the data was checked by the Shapiro–Wilk test. Mean differences between control cultures and cultures with isoflavones were assessed using the Student’s t-test.

3. Results and Discussion All strains grew at the maximum concentration of isoflavone glycosides (daidzin and genistin) and isoflavone aglycones (daidzein and genistein) used (MICs > 2048 µg mL−1). In contrast, susceptibility to equol ranged widely, from 16 µg mL−1 to 2048 µg mL−1 (Table 1). The strain most susceptible to equol was B. animalis subsp. animalis (MIC = 16 µg mL−1), while the most resistant strains belonged to the Gram-negative species E. coli and K. pneumoniae (MIC = 2048 µg mL−1). Eight strains, among which five species of lactobacilli, Slackia isoflavoniconvertens, and P. aeruginosa were found, showed an MIC of 1024 µg mL−1. The tested strains of Lactobacillus delbrueckii subsp. bulgaricus, Bacteroides fragilis, Bacteroides thetaiotaomicron, and Slackia equolifaciens showed moderate susceptibility to equol (MIC = 64 µg mL−1 for all). Overall, these results agree well with those reported in the literature, in which isoflavones lacking prenyl and hydroxyl groups at certain positions of the isoflavone ring structure—such as daidzin, genistin, daidzein and genistein—have shown no major antimicrobial activity [17]. Other studies describing isoflavones to have low antibacterial activity against Gram-negative bacteria have also been reported [16–18]. However, all these works had the aim of assessing isoflavones and their faecal-derived metabolites as potential antibacterial agents for counteracting the rise of antibiotic resistance among pathogens; this is why pathogenic species have been analysed so far [17]. In this work, a majority of strains under analysis were shown not to be inhibited by the tested compounds at concentrations higher than those reached at a physiological level (~200 µg mL−1 of intestinal content under usual treatment regimens; [11]). However, due to the large microbial complexity and diversity within the human gut [14,21], the response to isoflavones and their metabolites of members of bacterial groups others than those analysed in this study might be different. To determine whether isoflavones and equol could affect bacterial growth despite their high MIC values, the growth curves of 10 strains belonging to representative groups were investigated under specific culture conditions (see Section 2.1 and Table 1). As MICs of the isoflavone glycosides and isoflavone aglycones resulted identical, the former compounds were not tested in this assay. Since complete inhibition was not intended, the compounds to be assayed were added at a concentration below their MIC values (32 µg mL−1). Controls were prepared in which no phenolic compound was provided. Cultures were sampled hourly for the first 8 h of incubation and also after 24 h (at which time the maximum population size was attained). As expected, the growth kinetics recorded varied widely between bacterial groups (Figure 1). Except for slow-growing species (Sl. equolifaciens, Sl. flavoniconvertens, Faecalibacterium prausnitzii), standard deviation between assays was rather low for a microbial test, ranging from 0.03 to 0.23. Sl. equolifaciens did not appreciably grow during the first 8 h of incubation in any of the cultures. Broadly speaking, growth of the majority of the strains during these first 8 h (up to the beginning of the stationary phase in most cases) was very similar in the presence or absence of the test compounds, suggesting them to have no effect. Such was the case for Lactobacillus gasseri, Lactobacillus plantarum, Bifidobacterium longum, E. coli, and S. marcescens (Figure 1A–C,E,F, respectively). Growth curves similar to those of L. gasseri and L. plantarum were also obtained for Lactobacillus rhamnosus (data not shown). In contrast, cultures of F. prausnitzii, Lactococcus lactis subsp. lactis, and Bact. fragilis were inhibited by equol and even strongly

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Nutrients 2017, 9, 727 by genistein (Figure 1D,H,G, respectively). E. coli and S. marcescens grew better in the presence of isoflavones and equol than in the control cultures, although the difference was statistically significant for S. marcescens only (Figure 1F).

B A Lactobacillus gasseri Lactobacillus plantarum 3 3 2.5 2.5 2 2 1.5 1.5 1 1 0.5 0.5 0 0 0 2 4 6 8 0 2 4 6 8 C D 1 Bifidobacterium longum 0.2 Faecalibacterium prausnitzii

0.5 0.1 ** ** ** 0 0 0 2 4 6 8 0 2 4 6 8 E F Escherichia coli Serratia marcescens 1.5 1.5 * * *

Optical density nm 600 1 1

0.5 0.5

0 0 0 2 4 6 8 10 0 2 4 6 8 G H 3 Lactococcus lactis 3 Bacteroides fragilis 2.5 2.5 2 2 1.5 * 1.5 * * 1 * 1 * * 0.5 0.5 ** ** 0 0 * ** 0 2 4 6 8 0 2 4 6 8 Time (hours)

Figure 1. Growth curves of representative strains (A–H) as an average of the optical density (OD) measures of triplicate cultures in the presence of the soy isoflavone aglycones daidzein (in blue) and genistein (in red), and the daidzein-derived metabolite equol (in green) (all at 32 µg mL−1), as compared to a control without additives (in orange). Note that OD scale is different for different species. Mean values were compared by the Student’s t-test. Vertical bars show standard deviations (SD). Statistical significance: * p ≤ 0.05, ** p ≤ 0.01.

Deconjugation of isoflavone glycosides leads to the release of free glucose [6], which could then be used as a fuel. However, degradation of aglycones by certain species and their use as an energy source cannot be discarded. Indeed, beyond the conversion of daidzein into equol and genistein into 5-hydroxyequol, the catabolic profiling of soy aglycones and their derived metabolites by (intestinal) bacteria has scarcely been addressed [22,23]. As compared to the controls, daidzein causes small increases or decreases in the growth rate (µ) depending on the species (Table 2). µ also decreased moderately in some strains when either genistein

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Nutrients 2017, 9, 727 or equol was present in the culture medium, but increases were scored for some others. In accordance with the shape of their growth curves, the decrease in µ was particularly high for Bact. fragilis, L. lactis subsp. lactis, and Sl. equolifaciens. The enhanced growth rate of Lb. rhamnosus and S. marcescens in the presence of genistein and equol (Table 2) strongly suggests that somehow these species can degrade and use these compounds as an energy source. The catabolism of isoflavone glycosides, aglycones, and equol by strains of these species is currently underway. A particular case was F. prausnitzii. Equol and genistein inhibited growth of this species during the first 8 h of culture, but they both increased its µ value (calculated for this strain between 20 and 24 h). To examine the effects of aglycones and equol on the maximum optical density (OD) attained by cultures, this parameter was evaluated for the 10 selected strains at 24 h (Figure 2). Compared to the controls and reinforcing the observed changes in the µ, the presence of isoflavone aglycones or equol led to a lower final OD for some strains but higher for others. Of these changes, statistical significance was only found for the inhibition of Lb. gasseri by genistein, Bact. fragilis by both genistein and equol, and Sl. equolifaciens by all tested compounds.

Table 2. Growth rate of selected bacterial strains in cultures supplemented with daidzein, genistein, or equol at a final concentration of 32 µg mL−1 as compared to that in control cultures without isoflavone phenolics.

Strain/Culture Conditions Growth Rate a (µ) h−1 Species/Culture Conditions Growth Rate (µ) h−1 Lb. gasseri LMG 9203 b Bact. fragilis DSM 2151 d Control 0.747 Control 0.681 Daidzein 0.739 Daidzein 0.701 Genistein 0.738 Genistein 0.153 Equol 0.738 Equol 0.591 Lb. plantarum LMG 6907 b E. coli E-73 e Control 0.791 Control 0.772 Daidzein 0.755 Daidzein 0.775 Genistein 0.776 Genistein 0.820 Equol 0.722 Equol 0.787 Lb. rhamnosus LMG 6400 b S. marcescens S-54 e Control 0.867 Control 0.808 Daidzein 0.704 Daidzein 0.792 Genistein 0.942 Genistein 0.874 Equol 0.962 Equol 0.838 L. lactis subsp. lactis LMG 6890 c Sl. equolifaciens DSM 24851T Control 0.965 Control 0.238 Daidzein 0.736 Daidzein 0.174 Genistein 0.595 Genistein 0.121 Equol 0.799 Equol 0.153 B. longum subsp. longum LMG 13197 b F. prausnitzii DSM 17677 Control 0.581 Control 0.187 Daidzein 0.472 Daidzein 0.181 Genistein 0.516 Genistein 0.232 Equol 0.581 Equol 0.227 a The specific growth rate (µ) under the culture conditions was calculated as µ = Ln(N2/N1)/t2−t1, where N1 was the OD at t1 and N2 was the OD at t2. To calculate µ, a representative t1-t2 interval within the logarithmic growth phase of the cultures was selected. b de Man Rogosa and Sharpe (MRS) broth supplemented with 0.25% cysteine. c M17 broth supplemented with 1% glucose. d Gifu Anaerobic Medium (GAM) broth supplemented with 0.5% arginine. e Luria-Bertani (LB) broth.

The inhibitory activity of genistein against pathogens such as Staphylococcus aureus has been repeatedly reported [18,24]. As anticipated above, though the chemical structure of daidzein and genistein are very similar (except for the absence of an OH group in daidzein at position 5) [25], genistein inhibits DNA topoisomerease IV while daidzein does not [17], perhaps explaining its stronger antimicrobial action. It was surprising that the growth of Sl. equolifaciens was severely inhibited by all the test compounds; this and Sl. isoflavoniconvertens were the only equol-producing organisms among the tested bacteria. The equol used in this study was a racemic mixture of R- and S-enantiomers, while only the latter is produced endogenously in the

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Nutrients 2017, 9, 727 gut [8]. Therefore, as for some physiological effects [26], the antimicrobial action of the native equol might differ from that reported here. Moreover, soy isoflavones are metabolized into a vast array of chemically-related phenolic compounds [27,28] such as dihydrodaidzein, dihydrogenistein, tetrahydrodaidzein, O-desmethylangolensin (O-DMA), 5-hydroxyequol, and others [7,8,22], whose antimicrobial behaviour was not tested in this study. In addition, other phenolics, such as 4-ethylcatechol, 3-phenylpropionic acid, 3-hydroxyphenylacetic acid, and 4-hydroxy-5-phenylvaleric acid, have also been recorded to increase their faecal concentrations after isoflavone consumption [22]. Intermediate or end-product metabolites might have a range of biological properties, including antimicrobial activity. In fact, an antimicrobial effect of phenylacetic and phenylpropionic acids has already been reported, particularly against Gram-negative intestinal pathogens [29]. To provide a complete picture of how these compounds affect communities of gut bacteria, the antimicrobial properties of more isoflavone-derived phenolic compounds against representative gut bacteria should be examined. Besides, the use of culture-independent molecular methods to assess the quantification of bacterial growth (such as real-time quantitative PCR) could bring about more accurate results than those obtained by the culturing approach used in this work.

14

12

10

8 * Control 6 * Daidzein

4 ** Genistein Equol 2 ** ** ** 0

Figure 2. Final optical density (OD) at 600 nm after 24 h incubation of ten bacterial strains in the presence of 32 µg mL−1 of either daidzein, genistein, or equol, as compared to a control without phenolics. Standard deviation (SD) is indicated by vertical bars. Mean values were compared by the Student’s t-test. Statistical significance: * p ≤ 0.05, ** p ≤ 0.01.

4. Conclusions In conclusion, soy isoflavones and their metabolites are thought to have a range of beneficial health effects, which might be exerted through the modulation of bacterial populations in the human gut [1,3,30]. However, except for a few pathogens, studies examining the effects of these phenolic compounds on bacterial growth and metabolism have yet to be reported. To our knowledge, this is the first paper to report the resistance/susceptibility profiles of members of the commensal and beneficial bacterial communities of the human gut to isoflavones. The related parameters MIC, growth rate, and final growth estimate the competitiveness and fitness of bacteria in the presence of the compounds under study. Since isoflavone aglycones and equol can modify one or more of the variables examined, it might be concluded that when consumed either in food or in supplements, they may modify the total numbers and/or their relative proportions of specific bacterial communities in the gut.

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These modulatory effects on the intestinal bacterial populations might be associated with the beneficial properties attributed to soy consumption.

Acknowledgments: The study was partially supported by projects from the Spanish Ministry of Economy and Competitiveness (AGL-2014-57820-R) and Asturias Principality (GRUPIN14-137). L.G. and L.V. were supported by research contracts of the FPI Program from MINECO (BES-2012-062502 and BES-2015-072285, respectively). The skilful technical assistance of Paula Fernández is greatly acknowledged. Author Contributions: L.V. and L.G. performed most of the experiments and contributed to the discussion of the results. A.B.F. performed experiments and critically review the manuscript. B.M. provided material and human resources, drafted and reviewed the manuscript. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Messina, M. Soy and health update: Evaluation of the clinical and epidemiologic literature. Nutrients 2016, 8, 754. [CrossRef][PubMed] 2. Kang, J.; Badger, T.M.; Ronis, M.J.; Wu, X. Non-isoflavone phytochemicals in soy and their health effects. J. Agric. Food Chem. 2010, 58, 8119–8133. [CrossRef][PubMed] 3. Pilšáková, L.; Rieˇcanský, I.; Jagla, F. The physiological actions of isoflavone phytoestrogens. Physiol. Res. 2010, 59, 651–664. [PubMed] 4. De Cremoux, P.; This, P.; Leclercq, G.; Jacquot, Y. Controversies concerning the use of phytoestrogens in menopause management: Bioavailability and metabolism. Maturitas 2010, 65, 334–339. [CrossRef][PubMed] 5. Islam, M.A.; Bekele, R.; Vanden Berg, J.H.; Kuswanti, Y.; Thapa, O.; Soltani, S.; van Leeuwen, F.X.; Rietjens, I.M.; Murk, A.J. Deconjugation of soy isoflavone glucuronides needed for estrogenic activity. Toxicol. In Vitro 2015, 29, 706–715. [CrossRef][PubMed] 6. Landete, J.M.; Arqués, J.; Medina, M.; Gaya, P.; de Las Rivas, B.; Muñoz, R. Bioactivation of phytoestrogens: Intestinal bacteria and health. Crit. Rev. Food Sci. Nutr. 2016, 56, 1826–1843. [CrossRef][PubMed] 7. Kim, M.; Han, J. Isoflavone metabolism by human intestinal bacteria. Planta Med. 2016, 81, S1–S381. 8. Franke, A.A.; Lai, J.F.; Halm, B.M. Absortion, distribution, metabolism, and excretion of isoflavonoids after soy intake. Arch. Biochem. Biophys. 2014, 59, 24–28. [CrossRef][PubMed] 9. Clavel, T.; Fallani, M.; Lepage, P.; Levenez, F.; Mathey, J.; Rochet, V.; Sérézat, M.; Sutren, M.; Henderson, G.; Bennetau-Pelissero, C.; et al. Isoflavones and functional foods alter the dominant intestinal microbiota in postmenopausal women. J. Nutr. 2005, 135, 2786–2792. [PubMed] 10. Bolca, S.; Possemiers, S.; Herregat, A.; Huybrechts, I.; Heyerick, A.; De Vriese, S.; Verbruggen, M.; Depypere, H.; De Keukeleire, D.; Bracke, M.; et al. Microbial and dietary factors are associated with the equol producer phenotype in healthy postmenopausal women. J. Nutr. 2007, 137, 2242–2246. [PubMed] 11. Guadamuro, L.; Delgado, S.; Redruello, B.; Flórez, A.B.; Suárez, A.; Martínez-Camblor, P.; Mayo, B. Equol status and changes in faecal microbiota in menopausal women receiving long-term treatment for menopause symptoms with a soy-isoflavone concentrate. Front. Microbiol. 2015, 6, 777. [CrossRef][PubMed] 12. Nakatsu, C.H.; Arsmstrong, A.; Cavijo, A.P.; Martin, B.R.; Barnes, S.; Weaver, C.M. Fecal bacterial community changes associated with isoflavone metabolites in postmenopausal women after soy bar consumption. PLoS ONE 2014, 9, e108924. [CrossRef][PubMed] 13. Possemiers, S.; Bolca, S.; Eeckhaut, E.; Depypere, H.; Verstraete, W. Metabolism of isoflavones, lignans and prenylflavonoids by intestinal bacteria: Producer phenotyping and relation with intestinal community. FEMS Microbiol. Ecol. 2007, 61, 372–383. [CrossRef][PubMed] 14. Lozupone, C.A.; Stombaugh, J.I.; Gordon, J.I.; Jansson, J.K.; Knight, R. Diversity, stability and resilience of the human gut microbiota. Nature 2012, 489, 220–230. [CrossRef][PubMed] 15. Engels, C.; Schieber, A.; Gänzle, M.G. Inhibitory spectra and modes of antimicrobial action of gallotannins from Mango kernels (Mangifera indica L.). Appl. Environ. Microbiol. 2011, 77, 2215–2223. [CrossRef][PubMed] 16. Hummelova, J.; Rondevaldova, J.; Balstikova, A.; Lapcik, O.; Kokoska, L. The relationship between structure in vitro antibacterial activity of selected isoflavones and their metabolites with special focus on antistaphylococcal effect of demethyltexatin. Lett. Appl. Microbiol. 2014, 60, 242–247. [CrossRef][PubMed] 17. Mukne, A.P.; Viswanathan, V.; Phadatare, A.G. Structure pre-requisites for isoflavones as effective antibacterial agents. Pharmacogn. Rev. 2011, 5, 13–18. [CrossRef][PubMed]

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18. Verdrengh, M.; Collins, L.V.; Bergin, P.; Tarkowski, A. Phytoestrogen genistein as an anti-staphylococcal agent. Microbes. Infect. 2004, 6, 86–92. [CrossRef][PubMed] 19. CLSI (Clinical and Laboratory Standards Institute). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically. Standard M07-A10, 10th ed.; CLSI: Wayne, PA, USA, 2015. 20. CLSI (Clinical and Laboratory Standards Institute). Methods for Antimicrobial Susceptibility Testing of Anaerobic Bacteria. Standard M11-A8, 8th ed.; CLSI: Wayne, PA, USA, 2012. 21. Sankar, S.A.; Lagier, J.C.; Pontarotti, P.; Raoult, D.; Fournier, P.E. The human gut microbiome, a taxonomic conundrum. Syst. Appl. Microbiol. 2015, 38, 276–286. [CrossRef][PubMed] 22. Guadamuro, L.; Jiménez-Girón, A.M.; Delgado, S.; Flórez, A.B.; Suárez, A.; Martín-Álvarez, P.J.; Bartolomé, B.; Moreno-Arribas, M.V.; Mayo, B. Profiling of phenolic metabolites in feces from menopausal women after long-term isoflavone supplementation. J. Agric. Food. Chem. 2016, 64, 210–216. [CrossRef][PubMed] 23. Schwen, R.J.; Nguyen, L.; Jackson, R.L. Elucidation of the metabolic pathway of S-equol in rat, monkey and man. Food Chem. Toxicol. 2012, 50, 2074–2083. [CrossRef][PubMed] 24. Morán, A.; Gutiérrez, S.; Martínez-Blanco, H.; Ferrero, M.A.; Monteagudo-Mera, A.; Rodríguez-Aparicio, L.B. Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: A natural therapeutic strategy useful in the treatment and prevention of infections. Biofouling 2014, 30, 1175–1182. [CrossRef] [PubMed] 25. Del Rio, D.; Rodriguez-Mateos, A.; Spencer, J.P.; Tognolini, M.; Borges, G.; Crozier, A. Dietary (poly)phenolics in human health: Structures, bioavailability, and evidence of protective effects against chronic diseases. Antiox. Redox Signal. 2013, 18, 1818–1892. [CrossRef][PubMed] 26. Jackson, R.L.; Greiwe, J.S.; Schwen, R.J. Emerging evidence of the health benefits of S-equol, an estrogen receptor β agonist. Nutr. Rev. 2011, 69, 432–448. [CrossRef][PubMed] 27. Coldham, N.G.; Darby, C.; Hows, M.; King, L.J.; Zhang, A.Q.; Sauer, M.J. Comparative metabolism of genistin by human and rat gut microflora: Detection and identification of the end-products of metabolism. Xenobiotica 2002, 32, 45–62. [CrossRef][PubMed] 28. Setchell, K.D.; Brown, N.M.; Zhao, X.; Lindley, S.L.; Heubi, J.E.; King, E.C.; Messina, M.J. Soy isoflavone phase II metabolism differs between rodents and humans: Implications for the effect on breast cancer risk. Am. J. Clin. Nutr. 2011, 94, 1284–1294. [CrossRef][PubMed] 29. Lee, H.C.; Jenner, A.M.; Low, C.S.; Lee, Y.K. Effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota. Res. Microbiol. 2006, 157, 876–884. [CrossRef][PubMed] 30. Selma, M.V.; Espín, J.C.; Tomás-Barberán, F.A. Interaction between phenolics and gut microbiota: Role in human health. J. Agric. Food Chem. 2009, 57, 6485–6501. [CrossRef][PubMed]

© 2017 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 (http://creativecommons.org/licenses/by/4.0/).

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Article Metabolism of Soy Isoflavones by Intestinal Bacteria: Genome Analysis of an Adlercreutzia equolifaciens Strain That Does Not Produce Equol

Lucía Vázquez 1,2, Ana Belén Flórez 1,2 , Begoña Redruello 3 and Baltasar Mayo 1,2,* 1 Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), 33300 Villaviciosa, Spain; [email protected] (L.V.); abfl[email protected] (A.B.F.) 2 Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), 33011 Oviedo, Spain 3 Servicios Científico-Técnicos, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), 33300 Villaviciosa, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-985-89-33-45

 Received: 17 April 2020; Accepted: 20 June 2020; Published: 23 June 2020 

Abstract: Isoflavones are transformed in the gut into more estrogen-like compounds or into inactive molecules. However, neither the intestinal microbes nor the pathways leading to the synthesis of isoflavone-derived metabolites are fully known. In the present work, 73 fecal isolates from three women with an equol-producing phenotype were considered to harbor equol-related genes by qPCR. After typing, 57 different strains of different taxa were tested for their ability to act on the isoflavones daidzein and genistein. Strains producing small to moderate amounts of dihydrodaidzein and/or O-desmethylangolensin (O-DMA) from daidzein and dihydrogenistein from genistein were recorded. However, either alone or in several strain combinations, equol producers were not found, even though one of the strains, W18.34a (also known as IPLA37004), was identified as Adlercreutzia equolifaciens, a well-described equol-producing species. Analysis and comparison of A. equolifaciens W18.34a and A. equolifaciens DSM19450T (an equol producer bacterium) genome sequences suggested a deletion in the former involving a large part of the equol operon. Furthermore, genome comparison of A. equolifaciens and Asaccharobacter celatus (other equol-producing species) strains from databases indicated many of these also showed deletions within the equol operon. The present results contribute to our knowledge to the activity of gut bacteria on soy isoflavones.

Keywords: soy isoflavones; daidzein; genistein; equol; Adlercreutzia equolifaciens; intestinal microorganisms; fecal microbiota

1. Introduction The consumption of soy and soy-derived products correlates with better intestinal health, reduced menopause symptoms, and a smaller prevalence of hormone-mediated syndromes, cardiovascular disease, and cancer (for a recent review, see Zaheer et al. [1]). Soy has many biologically active compounds [2], but its beneficial health effects have been repeatedly attributed to its isoflavone content [3]. Isoflavones are polyphenols, the chemical structure of which resembles that of 17-β-oestradiol [4]; this invests these molecules with hormonal-like activity [5]. As recorded for other polyphenols, isoflavones also have antioxidant [6] and enzyme-inhibitory [7] properties. All of these features may contribute to their supposed health benefits. Dietary isoflavones are sequentially transformed into their active metabolites by cellular enzymes and enzymes from the gut microbiota [3]. Cellular and bacterial glycosyl hydrolases release isoflavone–aglycones from the isoflavone–glycosides present in plants [8]. Aglycones are

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Biomolecules 2020, 10, 950 more bioavailable and active than their glycoside counterparts [1,8], and can be further processed, undergoing dihydroxylation, reduction, breakage of the pyrone ring, or demethylation, etc., to produce compounds that either possess greater estrogenic activity (such as equol) or that are inactive (such as O-desmethylangolensin; O-DMA) [9]. However, while 80–90% of people can produce O-DMA, only 25–50% of humans produce equol [10–12]. Both O-DMA and equol are produced from the isoflavone daidzein via the exclusive action of intestinal bacteria [9]. Our knowledge of the species and pathways involved in the synthesis of the above and other isoflavone-derived metabolites remains scarce [5,13]. Improving is important since it may well be that only those persons able to produce certain metabolites such as equol may benefit fully from the consumption of soy or isoflavones [14,15]. In recent decades, several equol-producing strains of bacteria have been identified and characterized [16]. Most of them are anaerobic species belonging to the phylum Actinobacteria, class Coriobacteriia, family Eggerthellaceae [17]. Certainly, strains of Adlercreutzia equolifaciens [18], Asaccharobacter celatus [19], Enterorhabdus mucosicola [20], Slackia isoflavoniconvertens [21], Slackia equolifaciens [22], and other related bacteria have been reported to be equol producers. Equol biosynthesis in these strains takes place through dihydrodaidzein and tetrahydrodaidzein intermediates via a process involving three reductases [23–26]. Some equol-producing bacteria have also been shown to intervene in the conversion of genistein to 50-hydroxy-equol [27], a compound with a chemical structure similar to equol that might have comparable properties. However, it remains unknown whether other intestinal microbes are involved in isoflavone metabolism and equol formation [9], and this prevents us from developing strategies for modulating the endogenous production of isoflavone-bioactive metabolites via, for example, the use of prebiotics, probiotics, or other dietary supplements [28,29]. Furthermore, since bacterial equol and 50-hydroxy-equol producers are fastidious and extremely oxygen-susceptible, well-characterized strains of more manageable species might be better suited to the large-scale biotechnological production of these active derivatives. The aim of the present work was to contribute to the knowledge of isoflavone metabolism by intestinal bacteria. To this end, isolated colonies from fecal samples belonging to three women that produced equol were subjected to real-time quantitative PCR (qPCR) analysis, targeting the gene encoding the tetrahydrodaidzein reductase (tdr) required for equol biosynthesis. Presumptive positive isolates were then identified and typed by molecular methods, and the activity of different strains on daidzein and genistein was tested after culturing in the presence of these isoflavones. Finally, isoflavones and isoflavone-derived metabolites in the cultures were identified and quantified by ultra-high-performance liquid chromatography (UHPLC). In addition, a single recovered isolate of the species A. equolifaciens that did not produce equol from daidzein was subjected to genome sequencing and analysis, and its genome was then compared to those of closely related species available in databases.

2. Materials and Methods

2.1. Bacteria and Culture Conditions This study was approved by the Ethical Research Committee of Asturias Principality, Spain, with reference number 84/14. Intestinal bacteria were isolated from dilutions of fresh stool samples donated by three women with an equol-producing status (W3, W8, and W18) identified in a previous work [30]. Before sampling, volunteers signed a written informed consent. Variable amounts of dihydrogenistein, dihydrodaidzein, and equol from genistein and daidzein, respectively, were measured in fecal cultures inoculated with the same fecal samples utilized in this work as a source of microorganisms [30]. Dilutions of the samples were plated on Gifu Anaerobic Medium (GAM; Nissui, Tokyo, Japan) agar supplemented with 0.5% arginine (Merck, Darmstad, Germany), thus producing GAM-Arg plates, and incubated at 37 ◦C for up to five days under anoxic conditions (10% H2, 10% CO2, 80% N2) in a Mac500 work-station (Don Whitley, Bingley, UK). Though non-selective, GAM-Arg is suitable for growing equol producer strains of several species [30]. Colonies were collected daily and purified

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Biomolecules 2020, 10, 950 twice on the same plates. They were then inoculated into liquid GAM-Arg and stored at 80 C − ◦ with 15% glycerol (Merck). Adlercreutzia equolifaciens DSM 19450T, Asaccharobacter celatus DSM 18785T, Enterorhabdus mucosicola DSM 19490T, Slackia equolifaciens DSM 24851T, and Slackia isoflavoniconvertens DSM 22006T were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany), cultured under the above conditions, and used as equol-producing controls.

2.2. Quantitative Real-Time PCR (qPCR) Stored isolates were recovered on GAM-Arg agar plates and cell-free extracts from single colonies, obtained as described by Ruiz-Barba et al. [31] with minor modifications, and used in qPCR amplifications. Briefly, colonies were suspended in 100 µL of molecular-biology-grade water (Sigma-Aldrich, St. Louis, CA, USA), and subjected to heat treatment at 98 ◦C for 30 min. An equal volume of chloroform/isoamyl alcohol (24:1) (Sigma-Aldrich) was added and the cell suspensions vortexed for 5 s and then centrifuged at 16,000 g for 5 min. The upper aqueous phase was used as a source of DNA in the qPCR assays, × all performed in a 7500 Fast Real-Time PCR System running proprietary software v.2.0.4 (Applied Biosystems, Foster City, CA, USA). The qPCR was accomplished by using a primer pair targeting the tdr gene, which encodes a tetrahydrodaidzein reductase involved in equol production [30]. Briefly, reactions were performed in a final volume of 20 µL containing 10 µL of a 2xSYBR Green PCR Master Mix with ROX as a passive reference (Applied Biosystems), 900 nM of each primer, and 5 µL of cell-free extract. The standard amplification protocol consisted of an initial cycle at 95 ◦C for 10 min, followed by 40 cycles at 95 ◦C for 15 s, and 1 min at 60 ◦C. After amplification, the melting curves were analyzed and compared to those obtained with total DNA purified from the fecal samples of the women and that of the equol-producing bacterial controls.

2.3. Identification of Bacteria Isolates with a presumptive positive qPCR result were identified after isolation of their total DNA using the GenElute Bacterial Genomic DNA Kit (Sigma-Aldrich). To this end, the 16S rRNA gene was amplified using the universal oligonucleotide primers 27F (50-AGAGTTTGATCCTGGCTCAG-30) and 1492R (50-GGTTACCTTGTTACGACTT-30). The PCR conditions were as follows: one cycle at 95 ◦C for 5 min, 35 cycles at 94 ◦C for 30 s, 55 ◦C for 45 s, and 72 ◦C for 2 min, and a final extension cycle at 72 ◦C for 10 min. PCR products were subjected to electrophoresis in 2% agarose gels, stained with ethidium bromide (0.5 µg/mL), and visualized under UV light using a G Box Chemi XRQ gel doc system (Syngene International, Bangalore, India). Amplicons were then purified using GenElute PCR Clean-Up columns (Sigma-Aldrich) and sequenced at a sequencing service (Macrogen, Madrid, Spain). Sequences were then compared to those in the GenBank database using the BLAST+ software 2.10.0 version [32], and in the Ribosomal Database Project database Release 11 using the Classifier tool [33].

2.4. Typing of Isolates Isolates were genotyped according to their combined RAPD- and rep-PCR fingerprinting profiles using primer M13 (50-GAGGGTGGCGGTTCT-30) as reported by Rossetti and Giraffa [34], primer BoxA2R (50-ACGTGGTTTGAAGAGATTTTCBG-30) as reported by Koeuth et al. [35], and primer OPA18 (50-AGGTGACCGT-30) as reported by Mättö et al. [36]. PCR amplifications were independently performed in 25 µL volume reactions containing 12.5 µL MasterMix (Ampliqon), 5 µL of primer (10 µM), 3 µL of purified DNA, and molecular-grade water. The DNA amplification conditions were as follows: one cycle of 95 ◦C for 7min, 40 cycles of denaturation at 90 ◦C for 30 s, primer annealing for 1 min at 42 ◦C for M13, 40 ◦C for BoxA2R, or 32 ◦C for OPA18, an extension at 72 ◦C for 4 min, and a final extension step at 72 ◦C for 10 min. Amplicons were electrophoresed and visualized as above. GeneTools software v.4.03 (SynGene, Cambridge, UK) was used to compare and cluster the profiles using the unweighted pair group with the arithmetic mean (UPGMA) method. The similarity of patterns was expressed via simple matching (SM) coefficients. The results of triplicate typing analyses were 94% reproducible; profiles with 94% similarity were thus considered to be the same strain. ≥

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2.5. Detection and Quantification of Isoflavones and Isoflavone Metabolites Daidzein, genistein, and their derived metabolites dihydrodaidzein, dihydrogenistein, O-DMA, and equol were detected and quantified by UHPLC based on the method for isoflavone determination in urine samples reported by Redruello et al. [37]. Briefly, the control strains were independently cultured in GAM-Arg medium supplemented with 12.5–100 µM daidzein or genistein (LC Laboratories, Woburn, MA, USA). Furthermore, the selected strains were inoculated in pairs, triads, and tetrads and cultured with 100 µM of each isoflavone as above. After overnight incubation, cultures were centrifuged at 16,000 g for 2 min, and then filtered through a 0.2 µm polytetrafluoroethylene (PTFE) membrane (VWR, × Radnor, PA, USA). The culture supernatants were used directly in UHPLC analyses. Quantification was performed against calibration curves for isoflavone and isoflavone-derived standards obtained from a commercial source (LC Laboratories). In this work, the limit of quantification (LoQ) for the different compounds analyzed were, in µM, 6.25 for daidzein, genistein, and dihydrogenistein, 5.62 for O-DMA, 3.13 for dihydrodaidzein, and 3.12 for equol.

2.6. Genome Analysis of Adlercreutzia equolifaciens W18.34a DNA and deduced protein sequences from the genome of A. equolifaciens W18.34a (also known as IPLA 37004; [38]) were examined individually for homology against non-redundant DNA and protein databases using BLAST software (BLASTn and BLASTp, respectively) as above. To visualize the diversity and the evolutionary relationships between Coriobacteriia species, the genome sequences of type strains in GenBank were downloaded, aligned, and compared to that of W18.34a. A phylogenetic tree was created using the phylogenetic tree building service using PATRIC v.3.6.3 software [39] employing the “Codon Tree” workflow and the genome sequence of Bifidobacteirum longum subsp. infantis DSM 20,088 (GenBank NC_011593.1) as an outgroup. Briefly, alignments were performed against 100 shared protein sequences from the PATRIC global protein families (PGFams) using Muscle software [40]; nucleotide sequences were compared using the codonalign function in BioPython [41]. A concatenated alignment of all proteins and nucleotides was generated and visualized using Randomized Axelerated Maximum Likelihood (RAxML) [42] and FigTree software v. 1.4.3 (http: //tree.bio.ed.ac.uk/software/figtree/), respectively. Complementarily, genome sequences of all strains in GenBank belonging to A. equolifaciens and to the closely related species As. celatus were aligned and compared to the W18.34a genome using Mauve software v. 2.4.0 [43] and Vector NTI (Thermo Fisher Scientific, Waltham, MA, USA) programs.

3. Results More than 500 colonies from the dilutions of the fecal samples were screened by qPCR targeting the tdr gene (involved in the synthesis of equol). Analysis of the melting curves for 73 isolates suggested that these organisms might contain the target or a related gene. Despite this similarity in the melting curves, the Ct of the reactions was, in all cases, higher than 30, the limit of detection of the qPCR assay established in the previous work [30], suggesting this may represent a negative result. The molecular identification of the 73 isolates showed that they belonged to four distinct phyla, were grouped into 10 families, and belonged to 21 species-related taxa, of which the most abundant were Eggerthella lenta (19 isolates), Escherichia coli (17), Collinsella spp. (10), Bifidobacterium spp. (8), and Anaerococcus spp. (4) (Table 1). In addition, one of the isolates, W18.34a, was identified as A. equolifaciens, a well-known equol-producing species [18]. Under the experimental RAPD and rep-PCR typing conditions, 57 different strains were deemed detected among the 73 isolates (Figure S1).

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Table 1. Molecular identification by 16S rRNA gene sequencing of the isolates in this study.

Homology Phylum Class Family Species Isolate a % Identity to Sequence Escherichia coli W3.16 98.76 DQ857002.1 E. coli W3.21 99.00 CP026491.1 E. coli W8.4b 100.00 KR265473.1 E. coli W8.5 99.00 MF067516.1 E. coli W8.22b 99.00 CP019455.1 E. coli W8.24b 99.00 CP026491.1 E. coli W8.35b 99.00 KR265473.1 Proteobacteria γ-Proteobacteria Enterobacteriaceae E. coli W8.36b 99.31 MH782081.1 E. coli W8.39a 99.68 KY524291.1 E. coli W8.40 99.79 KY524291.1 E. coli W8.47 99.00 CP019455.1 E. coli W8.52b 99.00 KR265473.1 E. coli W8.53b 99.00 KP789326.1 E. coli W8.56c 98.41 KP244257.1 E. coli W18.17b 99.88 MH111527.1 E. coli W18.34b 99.00 CP019455.1 E. coli W18.19b 99.00 LC270191.1 Enterococcus durans W8.29 99.21 KY962884.1 Bacilli Enterococcaceae Enterococcus lactis W8.56b 99.68 MH431810.1 Anaerococcus spp. W3.10b 96.69 NR_114314.1 Anaerococcus spp. W8.44a 95.15 NR_114314.1 Anaerococcus vaginalis W3.18 98.43 NR_114314.1 A. vaginalis W8.44b 98.33 NR_041937.1 Firmicutes Bittarella massiliensis W18.24a 99.46 LN881596.1

Clostridia B. massiliensis W18.24b 99.55 LN881596.1 Finegoldia magna W8.4a 99.80 KC311751.1 Ruminococcaceae F. magna W8.35a 99.00 KR232883.1 Peptoniphilus gorbachii W8.22a 99.00 NR_115885.1 P. gorbachii W8.33 99.00 NR_115885.1 Streptococcaceae Streptococcus anginosus W8.21 99.00 JN787165.1 Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides vulgatus W8.28 99.00 CP013020.1 Porphyromonadaceae Parabacteroides spp. W8.41 95.56 CP022754.1 Actinomycetales Actinomycetaceae Actinomyces neuii W8.24a 99.56 NR_042429.1 Bifidobacterium adolescentis W8.34 99.30 KC174855.1 B. adolescentis W8.36a 99.00 KC174855.1 B. adolescentis W8.39b 99.00 LT223639.1 Bifidobacteriales Bifidobacteriaceae B. adolescentis W8.45 99.00 LT223639.1 B. adolescentis W8.54b 96.31 HM009032.1 Bifidobacterium animalis W3.3 99.31 MK561779.1 Bifidobacterium longum W8.17 99.00 KC174855.1 Actinobacteria B. longum W8.49a 98.00 HM009032.1 Collinsella aerofaciens W8.23 99.00 KP233323.1 C. aerofaciens W8.49b 99.00 KR232866.1 Collinsella massiliensis W18.17a 100.00 NR_144579.1 C. massiliensis W18.9 99.00 NR_144579.1

Coriobacteriia Coriobacteriaceae C. massiliensis W18.21a 99.00 NR_144579.1 C. massiliensis W18.21c 99.00 NR_144579.1 C. massiliensis W18.21d 99.00 NR_144579.1 C. massiliensis W18.25 99.00 NR_144579.1 C. massiliensis W18.29 99.00 NR_144579.1 Collinsella stercoris W18.1 97.80 KP233278.1

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Table 1. Cont.

Homology Phylum Class Family Species Isolate a % Identity to Sequence Adlercreutzia equolifaciens W18.34a 100.00 AB693938.1 Gordonibacter urolithinfaciens W18.23 99.00 NR_148261.1 G. urolithinfaciens W18.26 100.00 LT900217.1 Eggerthella lenta W3.2 99.00 KX683977.1 E. lenta W3.10a 99.00 KX683993.1 E. lenta W3.11 99.00 KX683977.1 E. lenta W3.12 99.00 KX683977.1 E. lenta W3.13 100.00 KX683977.1 E. lenta W3.14 99.00 KX683977.1 E. lenta W3.15 100.00 KX683977.1 Actinobacteria Eggerthellaceae E. lenta W8.15 98.00 KX683885.1 E. lenta W8.16 99.00 KX683992.1 E. lenta W8.27 99.00 KP944189.1 E. lenta W8.31 99.00 KP944189.1 E. lenta W8.42 99.00 JX104026.1 E. lenta W8.43 99.00 KX683992.1 E. lenta W8.52a 99.00 KP944190.1 E. lenta W8.53a 99.00 KX683992.1 E. lenta W8.54a 99.00 KP944189.1 E. lenta W8.56a 98.00 JX104026.1 E. lenta W18.18 99.00 KX683993.1 E. lenta W18.21b 100.00 KX683993.1 a Isolates were recovered from fresh stool samples of three women (W3, W8, and W18). In cultures, these fecal samples had been shown to produce dihydrodaidzein and equol from daidzein and dihydrogenistein from genistein [30].

To establish appropriate conditions for analyzing isoflavone metabolism, the control strains were incubated with varying concentrations of daidzein (12.5 to 100 µM) for 24 and 48 h (Table 2). Daidzein was rapidly used by all strains under most conditions; however, the synthesis of equol varied widely among the strains. At 24 h of incubation, S. isoflavoniconvertens DSM22006T and A. equolifaciens DSM 19450T transformed all daidzein into equol under all tested concentrations of daidzein, while S. equolifaciens DSM 24851T completed the transformation only when a concentration of 100 µM was used. The production of equol from daidzein by S. isoflavoniconvertens DSM22006T (Tables 2 and 3), and occasionally by A. equolifaciens DSM 19450T, reflected values higher than expected for the amount of daidzein added (Table 2). Smaller amounts of equol were always obtained with As. celatus DSM 18785T and E. mucosicola DSM 19490T. The concentration of equol was always higher at 24 than at 48 h, suggesting this compound is either further transformed or degraded by these strains under prolonged culturing. Based on these results, 100 µM isoflavone and 24 h incubation time were selected to test the fecal strains.

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Table 2. Production of equol from daidzein by type strains of equol-producing species.

Daidzein Supplementation (µM) 12.5 25 50 100 Equol-Producing Strain Daidzein-Derived Metabolite (µM) Daidzein Equol Daidzein Equol Daidzein Equol Daidzein Equol 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h A. equolifaciens DSM19450T - - 16.6 7.7 - - 33.6 13.5 - - 60.9 25.8 2.3 * - 126.2 53.4 As. celatus DSM18785T - - 3.1 3.3 nd - nd 6.3 - - 14.6 12.0 - - 27.7 27.8 E. mucosicola DSM19490T 2.1 * 1.4 * 4.5 2.0 * - - 17.9 6.7 5.1 * 1.4 * 26.2 10.5 60.3 36.2 nd nd S. equolifaciens DSM24851T - - 5.8 7.4 - - 14.4 13.3 1.1 * - 39.2 23.3 1.8 * - 110.0 47.8 S. isoflavoniconvertens DSM22006T - - 18.2 7.9 - - 37.0 14.3 - - 67.6 24.6 - - 149.0 55.0 -, not detected; nd, not done; asterisks denote samples with a concentration of the compounds below the LoQ.

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Table 3. Metabolism of daidzein and genistein by isolates and controls and the metabolites detected.

DZEN a (100 µM) GTEN (100 µM) Isolate DZEN D-DZEN O-DMA EQUOL GTEN D-GTEN Escherichia coli W3.16 61.2 - - - 64.9 4.8 * E. coli W3.21 61.9 - - - 60.0 4.3 * E. coli W8.4b 65.8 - - - 60.6 4.2 * E. coli W8.24b 85.1 - 3.3 * - 65.9 4.2 * E. coli W8.35b 76.3 - 2.8 * - 60.4 5.5 * E. coli W8.36b 73.2 1.5 * 6.8 - 68.6 - E. coli W8.40 73.2 3.0 * 3.0 * - 66.6 - E. coli W8.47 83.8 - 5.5 * - 62.8 - E. coli W8.53b 82.5 - 10.3 - 72.1 - E. coli W18.17b 87.5 - 11.2 - 72.0 - Enterococcus durans W8.29 85.1 - 3.7 * - 117.9 - Enterococcus lactis W8.56b 87.0 - 10.8 - 71.7 - Anaerococcus spp. W3.10b 93.6 - 10.9 - 75.5 - Anaerococcus spp. W8.44a 82.1 3.4 2.9 * - 62.9 - Anaerococcus vaginalis W3.18 62.0 - 2.3 * - 78.9 - Anaerococcus vaginalis W8.44b 79.8 - - - 48.2 - Bittarella massiliensis W18.24a 84.5 - 10.2 - 78.2 - Finegoldia magna W8.4a 61.2 - - - 55.7 2.7 * F. magna W8.35a 68.0 1.1 * 5.2 * - 62.6 - Peptoniphilus gorbachii W8.22a 69.5 3.9 10.0 - 40.3 4.3 * P. gorbachii W8.33 59.6 2.9 * 2.5 * - 40.3 - Streptococcus anginosus W8.21 99.6 - 2.9 * - 53.7 - Bacteroides vulgatus W8.28 73.1 - 3.7 * - 92.3 6.3 Parabacteroides spp. W8.41 72.3 - - - 68.6 - Actinomyces neuii W8.24a 53.5 2.8 * 5.1 * - 83.2 7.7 Bifidobacterium adolescentis W8.34 96.4 - - - 79.8 16.0 B. adolescentis W8.36a 110.5 - 3.4 * - 109.8 8.3 B. adolescentis W8.45 86.6 - 5.3 - 123.1 10.5 Bifidobacterium animalis W3.3 85.2 - 10.4 - 88.8 - Bifidobacterium longum W8.17 62.2 3.8 10.3 - 82.2 3.9 * B. longum W8.49a 136.1 - 2.3 * - 124.4 - Collinsella aerofaciens W8.23 109.1 3.7 11.7 - 84.6 6.1 * C. aerofaciens W8.49b 70.9 - - - 63.2 - Collinsella massiliensis W18.9 62.3 - 12.0 - 52.3 - C. massiliensis W18.17a 85.7 - 11.2 - 69.5 - C. massiliensis W18.21a 91.8 - 8.3 - 81.7 - C. massiliensis W18.21c 89.2 - 8.8 - 70.9 - C. massiliensis W18.21d 85.5 - 10.4 - 79.9 - C. massiliensis W18.25 84.6 - 12.0 - 76.5 - C. massiliensis W18.29 78.4 - - - 93.6 - Collinsella stercoris W18.1 87.8 - 9.4 - 81.2 - Adlercreutzia equolifaciens W18.34a 87.4 - 11.6 - 71.5 - Gordonibacter urolithinfaciens W18.23 76.9 - 10.8 - 73.3 - Eggerthella lenta W3.2 90.7 - 12.5 - 95.1 - E. lenta W3.10a 86.4 - 11.3 - 78.0 - E. lenta W3.11 89.1 - 11.6 - 80.4 - E. lenta W3.12 86.4 - 8.7 - 79.1 - E. lenta W3.13 88.3 - 10.7 - 81.9 - E. lenta W3.14 89.7 - 11.6 - 79.3 - E. lenta W8.15 66.3 3.9 9.8 - 58.6 6.4 E. lenta W8.16 65.7 3.3 8.7 - 58.4 3.4 * E. lenta W8.27 94.3 - 3.7 * - 82.1 4.4 * E. lenta W8.31 117.2 - - - 90.0 6.8 E. lenta W8.42 78.1 3.5 2.9 * - 62.2 - E. lenta W8.52a 97.9 - 8.9 - 63.5 - E. lenta W8.53a 87.3 - 9.0 - 73.0 - E. lenta W18.18 74.1 - 12.3 - 66.2 - Uninoculated medium - 98.1 5.8 1.4 0.2 - - 95.6 0.4 1.6 0.6 ± ± ± ± S. isoflavoniconvertens DSM 22006T - 1.6 0.2 - 164.3 13.4 55.3 19.0 ± ± a DZEN, daidzein; GTEN, genistein; D-DZEN y D-GTEN, dihydrodaidzein and dihydrogenistein, respectively; O-DMA, O-desmethylangolensin; -, not detected; asterisks indicate cultures with a concentration of the compound below the LoQ.

All 57 strains were assayed for isoflavone metabolism in GAM-Arg medium supplemented with either 100 µM of daidzein or genistein. Table 3 summarizes the results obtained. Between 40 and

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100% of the isoflavones added to the medium were recovered from the cultures as (correspondingly) unaltered daidzein or genistein. Isoflavone derived metabolites were clearly detected in some cultures even though the values obtained were occasionally below the limit of quantification. In the cultures with daidzein, low values of dihydrodaidzein (~3 µM) and/or O-DMA (~10 µM) were quantified in supernatants from some isolates of different bacterial lineages. The latter compound was mostly produced by members of the class Coriobacteriia, which includes species belonging to the families Coriobacteriaceae and Eggerthellaceae. Whenever a chromatographic peak was detected at the elution position of equol, the concentration was always below the LoQ for this compound (3.12 µM). This prompted all the analyzed strains to be deemed equol non-producers. As the original fecal samples produced equol but none of our isolates did, strains of different species were combined (in groups of two up to four) to test whether equol production was the result of complementary activities found in different microbes. Under the same culture conditions, no equol was detected when strain mixtures were grown together. Low levels of dihydrogenistein (3–7 µM) were detected in the supernatants of isolates from species such as Escherichia coli (seven strains) and E. lenta (four strains). The highest dihydrogenistein concentrations were detected in the supernatant of two Bifidobacterium adolescentis strains (10–16 µM). After incubation, the S. isoflavoniconvertens DSM22006T control strain converted about 20% of the genistein into dihydrogenistein. Surprisingly, strain W18.34a, identified as belonging to A. equolifaciens, produced some O-DMA from daidzein (about 10%), but did not produce any equol. This prompted the sequencing of its genome, recorded as IPLA37004 in the GenBank database (Assembly entry GCA_009874275.1) [38]. Phylogenomic analysis based on concatenated single-copy core-genome proteins and genes assigned W18.34a to a branch with A. equolifaciens and As. celatus strains (Figure 1): strains of the biotypes reported to produce equol. It should be noted, however, that these two species were described at around the same time [18,19], suggesting, as recently proposed, that they might still represent the same taxon [17]. Phylogenomic analysis of all A. equolifaciens and As. celatus strains in the NCBI database [44] comparing concatenated genome sequences reinforced this possibility (Figure 2).

Figure 1. Phylogenetic tree obtained by concatenated alignment of all proteins and genes from the genome sequence of Adlercreutzia equolifaciens W18.34a and other Coriboacteriia species and strains. The genome sequence of Bifidobacterium longum DSM 20088T was used as an outgroup.

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Figure 2. Phylogenetic relationships of Asaccharobacter celatus and Adlercreutzia equolifaciens strains (including W18.34a; IPLA 34007) obtained by comparison of their genome sequences.

In agreement with its equol-negative phenotype, no genes encoding reductases homologous to those involved in equol formation in A. equolifaciens, As. celatus, or Lactococcus garvieae were identified in the W18.34a genome. Comparison of DNA and deduced protein sequences from W18.34a to those of the equol-producing strain A. equolifaciens DSM19450T (GCA_000478885.1) showed the former to lack a region of about 11 kbp (Figure 3). This region contains a major part of the equol operon of A. equolifaciens DSM19450T [45]. In contrast, shared flanking ORFs upstream and downstream of the equol operon showed a deduced amino acid identity of 80–99% (Figure 3). Analysis of other genomes from NCBI showed that A. equolifaciens DSM19450T, A. equolifaciens KTCTC15235 (GCA_003428235.1), As. celatus DSM18785T (GCA_003726015.1), and As. celatus JCM14811 (GCA_003428485.1) harbored a complete equol operon in their genomes, while W18.34a, A. equolifaciens ResAG-91 (GCA_009755265.1), A. equolifaciens MGYG-HGUT-02480 (GCA_902387565.1), As. celatus AP38TSA (GCA_003340305.1), and As. celatus OB21 GAM11 (GCA_003340325.1) did not. The genetic organization of upstream and downstream ORFs around the equol gene cluster in several strains is shown in Figure 4. Furthermore, the assembled genome sequence of an uncultured Adlercreutzia spp. strain from a metagenomic project (SRA accession ERS2710141; GCA_900542605.1) also lacked equol genes, while maintaining highly homologous flanking DNA sequences to those of the above strains.

Figure 3. Diagram showing the genetic organization of upstream and downstream ORFs flanking the equol gene cluster in A. equolifaciens DSM19450T and A. equolifaciens W18.34a, and the percentage of amino acid identity of the deduced proteins encoded by shared genes. In green, genes coding for the well-known proteins involved in equol biosynthesis racemase (racemase), daidzein reductase (dzr), dihydrogenistein reductase (ddr), and tetrahydrodaidzein reductase (tdr); in yellow, other genes within the equol biosynthesis operon.

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Figure 4. Diagram showing the genetic organization of upstream and downstream ORFs within the equol gene cluster in several strains of A. equolifaciens and As. celatus. In green, genes coding for well-known proteins involved in equol biosynthesis: racemase (racemase), daidzein reductase (dzr), dihydrogenistein reductase (ddr), and tetrahydrodaidzein reductase (tdr); in yellow, genes coding proteins with activity in daidzein metabolism; in light brown, conserved genes along all analyzed strains; in dark brown, red, pink, and purple, genes present in certain strains but not in others; and in pale blue, strain-specific genes. A. equolifaciens DSM19450T and As. celatus DSM18785T have been reported to be equol producers, while A. equolifaciens W18.34a does not produce equol.

4. Discussion The high isoflavone consumption of Asian populations (compared to that of Westerners) has been epidemiologically associated with less severe menopause symptoms and a lower prevalence of cardiovascular diseases, osteoporosis, and cancer [1]. However, the actual metabolite(s) that impart these beneficial health effects, the target tissue(s), and the underlying signaling cascade(s) have yet to be discovered [46]. Although it is well established that the synthesis of equol from daidzein is carried out exclusively by certain members of the gut microbiota, the actual microbes involved are not well-identified [1,5,16]. In this work, three fecal samples, which have proven to produce equol [30], were used as a source of isoflavone-acting microbes. Plating on GAM-Arg agar has been shown to be appropriate for isolating strains of the majority and subdominant bacterial populations from feces [47] including members of the class Coriobacteriia, family Eggerthellaceae, where most equol producers of intestinal origin are currently allocated [17]. The qPCR strategy targeting the tdr gene, however, did not result in the identification of any equol-producing isolate, even though 73 of them were initially considered as possibly positive. In agreement with their obligate anaerobic nature, most intestinal species possess large numbers of reductase-encoding genes [48]. As an example, 166 ORFs have been annotated as reductase-encoding genes in the genome of A. equolifaciens W18.34a [38]. Some of these might have regions of similarity to those of the tdr genes used in the design of the present degenerate primers, which led to unspecific amplification. If isolation of equol producers is the goal, a different approach and/or a higher testing effort would be required. As reported elsewhere [49], equol production might also result from the complementary activity of two or more microbes, which will complicate the identification of equol producers. Daidzein and genistein were partially transformed by many isolates. For daidzein, small amounts of dihydrodaidzein and moderate amounts of O-DMA were quantified in the culture supernatants of several strains. The activity of one or more of the (unspecific) reductases above-mentioned might be responsible for the formation of small amounts of these isoflavone-derived metabolites. However, under the present study conditions, no strain tested produced equol. As the donor fecal cultures produced equol [30], the production of this compound was deemed feasible if strains from different species were combined in groups of two, three, or four, but this was not the case, which indicates that those tested

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Biomolecules 2020, 10, 950 had no complementary activities that would lead to equol synthesis. Similarly, moderate amounts of dihydrogenistein were detected in the supernatant of some strains when cultured with genistein (some of which did not produce dihydrodaidzein from daidzein). The control strain S. isoflavoniconvertens DSM 22006T has been reported to be a 5-hydroxy equol producer [23]. However, the lack of an appropriate commercial standard hindered the determination in this study of this isoflavone-derivative. As previously reported and discussed, isoflavones may be transformed into a variety of unidentified metabolites [30,50]. In the absence of standards, identification of some of those that are known to be produced (e.g., 5-hydroxy-equol, 5-hydroxy-dehydroequol) may require high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS) analysis and/or sophisticated chirality studies natural and chemically synthesized substances [51,52]. Apart from the deglycosilation step [8,53], our knowledge of the microbes and molecular pathways involved in isoflavone metabolism is still limited [54]. Most intestinal bacteria acting on isoflavones belong to the family Eggerthellaceae [17]. However, whether other bacterial species in the gut participate in the metabolism of isoflavones and the formation of equol and/or 5-hydroxy-equol remains unknown. In addition, the issue of whether the production of these compounds by the Eggerthellaceae is a family-, species-, or strain-specific trait is yet to be resolved [16]. It was therefore surprising to identify an A. equolifaciens strain from the feces of an equol-producing woman that did not produce equol. Genome analysis of this strain revealed a major part of the equol gene cluster to be absent. In the type strain of this species, A. equolifaciens DSM19450T, this cluster is composed of 10–13 ORFs organized into an operon-like structure [45]. Conceivably, the absence of equol-related genes is the cause of the equol non-producing phenotype in A. equolifaciens W18.34a. Analysis of the available genomes in the National Center for Biotechnology Information (NCBI) database identified more or less equal numbers of A. equolifaciens and As. celatus strains with and without most of the equol-related genes. All equol-producing strains harbor equol-associated genes, particularly those coding for a racemase and three reductases, namely daidzein reductase, dihydrodaidzein reductase, and tetrahydrodaidzein reductase [23–26,55–57]. However, with the exception of A. equolifaciens W18.34a, nothing is known about the equol phenotype of strains lacking genes within the equol operon. Whether there has been a deletion in strains lacking the locus or a gain-of-function in equol producers is currently a matter of speculation. However, the fact that in all strains lacking genes of the equol operon both upstream and downstream genes conserve a high degree of linearity and their deduced proteins show strong amino acid identity argues for the deletion of genes in certain strains. This suggests that the equol-producing phenotype does not currently provide a selective advantage in the human intestine to bacteria, thus leading to a loss of metabolic function. This agrees well with only a small percentage of humans (depending on dietary habits and human community) carrying equol-producing microbes in their gut [11,12,58], while all the animals tested so far are able to produce equol in response to soy or daidzein consumption [59]. The presence in the human gut of equol producing and equol non-producing Eggerthellaceae is further strengthened by the repeated counting of similar numbers of equol-related taxa in fecal samples from equol producers and non-producers [30,60–63]. As a consequence, determining the equol-producing status in humans based on taxonomic criteria alone is unreliable [63,64].

5. Conclusions In this study, strains of several bacterial species from human feces able to produce small to moderate amounts of dihydrodaidzein and O-DMA from daidzein and of dihydrogenistein from genistein were detected. No association was seen between the formation of dihydrodaidzein from daidzein and that of dihydrogenistein from genistein, although some strains produced both isoflavone derivatives. None of the strains tested produced equol from daidzein, even though isolate W18.34a (IPLA 37004) was identified as A. equolifaciens. Other bacterium or a bacterial consortium not isolated in the present work may be responsible for the equol-producing phenotype of the women who supplied the fecal samples. Genome analyses of W18.34a suggested the deletion of most of the genes in the

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Biomolecules 2020, 10, 950 equol operon in this strain, and in others of A. equolifaciens and As. celatus. This argues in favor of the coexistence of equol-producing and non-producing bacterial strains in the human gut, suggesting the former phenotype does not provide a selective advantage. However, more studies are still required to unravel the complex relationships of isoflavones and components of the gut microbiota with emphasis on the synthesis of physiologically-active derived molecules.

Supplementary Materials: The following are available online at http://www.mdpi.com/2218-273X/10/6/950/s1, Figure S1: Dendogram of similarity of the combined typing profiles obtained with primers OPA18, M13, and BoxA2R expressed by the Simple Matching (SM) coefficient. Clustering was performed by the unweighted pair group method using arithmetic averages (UPGMA). The dotted line indicates the repeatability of the combined typing method (~94%). Author Contributions: Conceptualization, B.M. and A.B.F.; Methodology, A.B.F. and B.R.; Formal analysis, B.M., A.B.F., B.R., and L.V.; Investigation, A.B.F. and L.V.; Resources, B.M.; Supervision, B.M. and A.B.F., Writing—original draft preparation, B.M.; Writing—review and editing, B.M., A.B.F., and L.V. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by projects from the Spanish Ministry of Economy and Competitiveness (MINECO) (AGL2014-57820-R) and the Principality of Asturias (IDI/2018/000114). L.V. was supported by a research contract from the FPI Program from MINECO (BES-2015-072285). Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

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12. Song, K.B.; Atkinson, C.; Frankenfeld, C.L.; Jokela, T.; Wähälä, K.; Thomas, W.K.; Lampe, J.W. Prevalence of daidzein-metabolizing phenotypes differs between Caucasian and Korean American women and girls. J. Nutr. 2006, 136, 1347–1351. [CrossRef][PubMed] 13. Kemperman, R.A.; Bolca, S.; Roger, L.C.; Vaughan, E.E. Novel approaches for analysing gut microbes and dietary polyphenols: Challenges and opportunities. Microbiology 2010, 156, 3224–3231. [CrossRef][PubMed] 14. Newton, K.M.; Reed, S.D.; Uchiyama, S.; Qu, C.; Ueno, T.; Iwashita, S.; Gunderson, G.; Fuller, S.; Lampe, J.W. A cross-sectional study of equol producer status and self-reported vasomotor symptoms. Menopause 2015, 22, 489–495. [CrossRef][PubMed] 15. Wong, J.M.; Kendall, C.W.; Marchie, A.; Liu, Z.; Vidgen, E.; Holmes, C.; Jackson, C.J.; Josse, R.G.; Pencharz, P.B.; Rao, A.V.; et al. Equol status and blood lipid profile in hyperlipidemia after consumption of diets containing soy foods. Am. J. Clin. Nutr. 2012, 95, 564–571. [CrossRef] 16. Mayo, B.; Vázquez, L.; Flórez, A.B. Equol: A bacterial metabolite from the daidzein isoflavone and its presumed beneficial health effects. Nutrients 2019, 11, 2231. [CrossRef] 17. Salam, N.; Jiao, J.Y.; Zhang, X.T.; Li, W.J. Updat0e on the classification of higher ranks in the phylum Actinobacteria. Int. J. Syst. Evol. Microbiol. 2020, 70, 1331–1355. [CrossRef] 18. Maruo, T.; Sakamoto, M.; Ito, C.; Toda, T.; Benno, Y. Adlercreutzia equolifaciens gen. nov., sp. nov., an equol-producing bacterium isolated from human faeces, and emended description of the genus Eggerthella. Int. J. Syst. Evol. Microbiol. 2008, 58, 1221–1227. [CrossRef] 19. Minamida, K.; Ota, K.; Nishimukai, M.; Tanaka, M.; Abe, A.; Sone, T.; Tomita, F.; Hara, H.; Asano, K. Asaccharobacter celatus gen. nov., sp. nov., isolated from rat caecum. Int. J. Syst. Evol. Microbiol. 2008, 58, 1238–1240. [CrossRef] 20. Clavel, T.; Charrier, C.; Braune, A.; Wenning, M.; Blaut, M.; Haller, D. Isolation of bacteria from the ileal mucosa of TNFdeltaARE mice and description of Enterorhabdus mucosicola gen. nov., sp. nov. Int. J. Syst. Evol. Microbiol. 2009, 59, 1805–1812. [CrossRef] 21. Matthies, A.; Blaut, M.; Braune, A. Isolation of a human intestinal bacterium capable of daidzein and genistein conversion. Appl. Environ. Microbiol. 2009, 75, 1740–1744. [CrossRef] 22. Jin, J.S.; Kitahara, M.; Sakamoto, M.; Hattori, M.; Benno, Y. Slackia equolifaciens sp. nov., a human intestinal bacterium capable of producing equol. Int. J. Syst. Evol. Microbiol. 2010, 60, 1721–1724. [CrossRef][PubMed] 23. Schröder, C.; Matthies, A.; Engst, W.; Blaut, M.; Braune, A. Identification and expression of genes involved in the conversion of daidzein and genistein by the equol-forming bacterium Slackia isoflavoniconvertens. Appl. Environ. Microbiol. 2013, 79, 3494–3502. [CrossRef][PubMed] 24. Tsuji, H.; Moriyama, K.; Nomoto, K.; Akaza, H. Identification of an enzyme system for daidzein-to-equol conversion in Slackia sp. strain NATTS. Appl. Environ. Microbiol. 2012, 78, 1228–1236. [CrossRef][PubMed] 25. Shimada, Y.; Yasuda, S.; Takahashi, M.; Hayashi, T.; Miyazawa, N.; Sato, I.; Abiru, Y.; Uchiyama, S.; Hishigaki, H. Cloning and expression of a novel NADP(H)-dependent daidzein reductase, an enzyme involved in the metabolism of daidzein, from equol-producing Lactococcus strain 20–92. Appl. Environ. Microbiol. 2010, 76, 5892–5901. [CrossRef] 26. Shimada, Y.; Takahashi, M.; Miyazawa, N.; Ohtani, T.; Abiru, Y.; Uchiyama, S.; Hishigaki, H. Identification of two novel reductases involved in equol biosynthesis in Lactococcus strain 20–92. J. Mol. Microbiol. Biotechnol. 2011, 2, 160–172. [CrossRef][PubMed] 27. Matthies, A.; Loh, G.; Blaut, M.; Braune, A. Daidzein and genistein are converted to equol and 5-hydroxy-equol by human intestinal Slackia isoflavoniconvertens in gnotobiotic rats. J. Nutr. 2012, 142, 40–66. [CrossRef] 28. Martinez, I.; Kim, J.; Duffy, P.R.; Schlegel, V.L.; Walter, J. Resistant starches types 2 and 4 have differential effects on the composition of the fecal microbiota in human subjects. PLoS ONE 2010, 5, e15046. [CrossRef] 29. Jumpertz, R.; Le, D.S.; Turnbaugh, P.J.; Trinidad, C.; Bogardus, C.; Gordon, J.I.; Krakoff, J. Energy-balance studies reveal associations between gut microbes, caloric load, and nutrient absorption in humans. Am. J. Clin. Nutr. 2011, 94, 58–65. [CrossRef] 30. Vázquez, L.; Guadamuro, L.; Giganto, F.; Mayo, B.; Flórez, A.B. Development and use of a real-time quantitative PCR method for detecting and quantifying equol-producing bacteria in human faecal samples and slurry cultures. Front. Microbiol. 2017, 8, 1155. [CrossRef] 31. Ruiz-Barba, J.L.; Maldonado, A.; Jiménez-Díaz, R. Small-scale total DNA extraction from bacteria and yeast for PCR applications. Anal. Biochem. 2005, 347, 333–335. [CrossRef]

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© 2020 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 (http://creativecommons.org/licenses/by/4.0/).

96 CAPÍTULO 2 MATERIAL SUPLEMENTARIO

A) Family Enterobacteriaceae B) Family Enterococcaceae

53 62 72 81 91 100 25 40 55 70 85 100 W 8.39a W 8.29 W 8.40 W 8.56 W 8.53b F) Family Eggerthellaceae W 3.10b W 8.56c 43 54 66 77 89 100 W 8.44a W 8.36b W 8.42 W 3.18 W 8.35b W 8.43 W 8.47 W 8.44b W 8.27 W 8.52b W 8.31 W 8.24b C) Family Ruminococcaceae W 8.4b W 8.52a 15 32 49 66 83 100 W 8.5 W 8.53a W 18.24a W 8.22b W 8.54a W 18.24b W 18.17b W 8.56a W 18.34b W 8.35a W 3.10a W 18.19b W 8.22a W 3.2 W 3.16 W 8.33 W 3.21 W 3.13 W 8.4a W 3.11 W 3.12 D) Family Bifidobacteriaceae E) Family Coriobacteriaceae W 3.14 11 29 47 64 82 100 23 38 54 69 85 100 W 3.15 W 8.36a W 8.23 W 8.15 W 8.39b W 8.49b W 8.16 W 18.1 W 18.18 W 8.45 W 18.17a W 18.21b W 8.54b W 18.25

W 8.34 W 18.9 28 43 57 71 86 100 W 18.21a W 18.23 W 8.49a W 18.21d W 18.26 W 8.17 W 18.21c W 18.34a W 3.3 W 18.29

Figure S1.- Dendogram of similarity of the combined typing profiles obtained with primers OPA18, M13, and BoxA2R expressed by the Simple Matching (SM) coefficient. Clustering was performed by the unweighted pair group method using arithmetic averages (UPGMA). The dotted line indicates the repeatability of the combined typing method (~94%).

97 CAPÍTULO 2 GENOME SEQUENCES crossm

Draft Genome Sequence of Adlercreutzia equolifaciens IPLA 37004, a Human Intestinal Strain That Does Not Produce Equol from Daidzein

Lucía Vázquez,a,b Ana Belén Flórez,a,b Baltasar Mayoa,b aDepartamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Asturias, Spain bInstituto de Investigación Sanitaria del Principado de Asturias (ISPA), Oviedo, Asturias, Spain

ABSTRACT Equol is an intestinal bacterial metabolite derived from the isoflavone daidzein and has beneficial health effects. Most equol producers belong to the fam- ily Coriobacteriaceae, which includes species such as Adlercreutzia equolifaciens. Here, we report the draft genome sequence of A. equolifaciens IPLA 37004, a human iso- late that does not produce equol.

mong isoflavones and their metabolites, the bacterial daidzein-derived compound A(S)-(Ϫ)-equol has the strongest estrogenic activity (1) and antioxidant action (2), by which it can positively influence human health (3). However, only 30 to 60% of humans harbor equol-producing microbes in their intestines. Most equol-producing bacteria belong to the family Coriobacteriaceae of the phylum Actinobacteria, which includes species such as Adlercreutzia equolifaciens, Asaccharobacter celatus, Enterorhabdus mu- cosicola, Slackia isoflavoniconvertens, and Slackia equolifaciens (3). However, whether equol production is a family-, species-, or strain-specific characteristic is not yet known. Strain IPLA 37004 was isolated from fecal samples from a woman with an equol- producing microbiota, using agar plates of Gifu anaerobic medium (GAM) (Nissui) supplemented with 5 g literϪ1 arginine (GAM-Arg; Merck) and cultivation under strict anaerobic conditions. Sequencing and sequence comparison of the 16S rRNA gene of IPLA 37004 identified the strain as A. equolifaciens. Surprisingly, the strain did not produce equol when cultured in GAM-Arg with isoflavones, daidzein, or dihydrodaid- zein. To get insights regarding its non-equol-producing phenotype, the genome of A. equolifaciens IPLA 37004 was sequenced, and its sequence was analyzed. After growth of the strain in GAM-Arg, total genomic DNA from IPLA 37004 was extracted and purified using the DNeasy blood and tissue kit (Qiagen), as suggested by the manufacturer. For genome sequencing, a library was constructed using SPRIworks Citation Vázquez L, Flórez AB, Mayo B. 2020. Draft genome sequence of Adlercreutzia fragment library system I (Beckman Coulter), according to the manufacturer’s instruc- equolifaciens IPLA 37004, a human intestinal tions. Paired-end sequencing (2 ϫ 150-bp reads) was performed at Eurofins Genomics strain that does not produce equol from on a HiSeq sequencer (Illumina) (sequence mode, NovaSeq 6000; S2 flow cell; paired- daidzein. Microbiol Resour Announc ϫ 9:e01537-19. https://doi.org/10.1128/MRA end 150-bp reads; Xp flowchart). The average genome coverage approached 100 , .01537-19. yielding a total of 5,568,276 bp of adapter-free and quality-filtered reads after the use Editor David Rasko, University of Maryland of Trimmomatic (4) and FastQC (5) software packages. De novo assembly of quality- School of Medicine filtered nontrimmed reads in contigs was accomplished using SPAdes v3.6.2 (6), setting Copyright © 2020 Vázquez et al. This is an the parameters with a k value of 125 or 127 and only-assembler. Then, the NCBI open-access article distributed under the terms of the Creative Commons Attribution 4.0 Prokaryotic Genome Annotation Pipeline (7) was used to predict and annotate the open International license. reading frames. Whole-genome comparisons were achieved by using the multiple Address correspondence to Baltasar Mayo, genome alignment software package Mauve v2.3.1 (8). Finally, BLAST v2.10.0 searches [email protected]. (BLASTp suite, NCBI) were used to determine homology between proteins of different Received 16 December 2019 Accepted 1 February 2020 strains. Published 20 February 2020 The genome of A. equolifaciens IPLA 37004 consisted of 2,664,741 bp in 169 contigs

Volume 9 Issue 8 e01537-19 98 mra.asm.org CAPÍTULO 2 Vázquez et al. longer than 200 bp, with a GϩC content of 63.7%. The maximum contig length was

325,998 bp, and the N50 and N90 values were 101,827 and 25,297 bp, respectively. A total of 2,310 genes were identified in the IPLA 37004 genome, including 2,251 coding sequences, of which 2,198 encoded complete proteins and 53 were identified as pseudogenes. In addition, 59 genes encoded RNA molecules, including 8 rRNAs, 48 tRNAs, and 3 noncoding RNAs. Genes encoding reductases homologous to those of A. equolifaciens DSM 19450T involved in equol formation (9) were not detected in the IPLA 37004 genome. Whole-genome comparison of DSM 19450T and IPLA 37004 identified shared flanking genes upstream and downstream of the equol locus in A. equolifaciens DSM 19450T. The deduced amino acids of the shared genes showed an identity range of 80 to 99%. The gene cluster of DSM 19450T encompassed a region of about 11 kbp, which was absent in IPLA 37004. Data availability. The genome sequence of A. equolifaciens IPLA 37004 was depos- ited in GenBank under accession number VJNE00000000. The fastq files of the raw reads were deposited in the NCBI SRA under accession number SRR9657807.

ACKNOWLEDGMENTS This study was supported by projects from MINECO (grant AGL-2014-57820-R) and Asturias Principality (grant IDI/2018/000114).

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Volume 9 Issue 8 e01537-19 99 mra.asm.org CAPÍTULO 3

CAPÍTULO 3

Caracterización de la producción de equol en muestras fecales y bacterias productoras con el fin de maximizar su síntesis endógena y biotecnológica.

En este capítulo de la Tesis se muestran los resultados de tres artículos: en el primero, se utiliza un modelo intestinal dinámico capaz de simular las condiciones del colon proximal humano (TIM-2) para evaluar el efecto de la dieta en la producción de equol en cultivos fecales; en el segundo, se caracterizan los elementos génicos involucrados en la formación de equol y los mecanismos implicados en su regulación en la cepa A. equolifaciens DSM19450T; y en un artículo final, se diseña un segmento de ADN sintético que incluye los elementos esenciales de la maquinaria genética necesaria para la producción de equol, se clona en la bacteria modelo Escherichia coli DH10B y, finalmente, se transfiere a bacterias ácido-lácticas de interés alimentario.

. Artículo VI.- Vázquez, L., Flórez, A. B., Verbruggen, S., Redruello, B., Verhoeven, J., Venema, K. y Mayo, B. (2020). Modulation of equol production via different dietary regimens in an artificial model of the human colon. Journal of Functional Foods, 66, 103819. doi:10.1016/j.jff.2020.103819.

. Artículo VII.- Flórez, A. B., Vázquez, L., Rodríguez, J., Redruello, B. y Mayo, B. (2019). Transcriptional regulation of the equol biosynthesis gene cluster in Adlercreutzia equolifaciens DSM19450T. Nutrients 11(5), 993. doi:10.3390/nu11050993.

. Manuscrito VIII.- Cloning and expression of equol genes from Adlercreutzia equolifaciens in Escherichia coli and lactic acid bacteria.

En cuanto al efecto de la dieta, mediante el estudio efectuado con el TIM-2 se observó que bajo un régimen alimenticio enriquecido en carbohidratos o proteínas las poblaciones mayoritarias del TGI de mujeres productoras de equol no experimentan grandes cambios, al menos en un corto periodo de tiempo. Por el contrario, la producción de equol variaba mucho en función del tipo de dieta. Con la dieta rica en carbohidratos se duplicó la producción de equol endógeno, mientras que con la dieta rica en proteínas esta se redujo prácticamente a la mitad. En conclusión, los ensayos del TIM-2 indican que la dieta podría ser un factor de utilidad para la modulación de las poblaciones intestinales y para el incremento de la producción endógena de equol.

100 CAPÍTULO 3

A. equolifaciens DSM19450T es capaz de metabolizar completamente la daidzeína presente en el medio de cultivo tras 10 h de incubación. Sin embargo, solo una tercera parte de la daidzeína se transforma en dihidrodaidzeína y, posteriormente, en equol. El análisis transcripcional reveló la sobreexpresión de 13 genes contiguos cuando la daidzeína estaba presente en el medio de cultivo, lo que sugiere que esta isoflavona modula la expresión de todos estos genes. La sobreexpresión varía en función del gen y de la concentración de daidzeína, pero se observaron cuatro patrones de expresión diferencial, cada uno bajo el control de secuencias promotoras y terminadoras. La amplificación mediante RT-PCR de todas las regiones intergénicas reveló que el operón se trascribe como una única molécula de ARN mensajero.

En un trabajo posterior, ADN sintético incluyendo los genes tdr, ddr y dzr que codifican para las reductasas, junto con el gen de la racemasa, se clonó en E. coli. Las bacterias recombinantes eran capaces de producir equol a partir de daidzeína, confirmando que los genes de A. equolifaciens DSM19450T eran funcionales en este huésped heterólogo. Posteriormente, los genes de interés se transfirieron a un vector bifuncional E. coli-BAL (pIL252) y se introdujeron en cepas de Lactococcus lactis y Lactobacillus casei mediante electroporación. En esta última especie se observó también la producción de equol a partir de su precursor, dihidrodaidzeína. Estos resultados indican que es posible expresar la maquinaria genética de producción de equol en BAL, si bien serán necesarios nuevos trabajos para optimizar y maximizar su producción por bacterias de este grupo.

El conocimiento obtenido en estos trabajos resultará clave para, en el futuro, incrementar la producción endógena de equol o maximizar la producción biotecnológica de este compuesto.

101 CAPÍTULO 3 Journal of Functional Foods 66 (2020) 103819

Contents lists available at ScienceDirect

Journal of Functional Foods

journal homepage: www.elsevier.com/locate/jff

Modulation of equol production via different dietary regimens in an artificial model of the human colon T

Lucía Vázqueza,b,c, Ana Belén Flóreza,b, Sanne Verbruggenc, Begoña Redruellod, ⁎ Jessica Verhoevenc, Koen Venemac, Baltasar Mayoa,b, a Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares, s/n, 33300 Villaviciosa, Spain b Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain c Centre for Healthy Eating & Food Innovation-Maastricht University, Campus Venlo, St. Jansweg 20, 5928 RC Venlo, the Netherlands d Servicios Científico-Técnicos, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares, s/n, 33300 Villaviciosa, Spain

ARTICLE INFO ABSTRACT

Keywords: In order to find dietary conditions favouring endogenous equol biosynthesis, a pooled faecal homogenate from Soy isoflavones equol-producing women was used to inoculate the TIM-2 artificial model of the human proximal colon. The Daidzein model was fuelled with control diets not supplemented (C) or supplemented (C-ISO) with isoflavones, and two Equol isoflavone-containing diets rich in carbohydrate (CH-ISO) or protein (PR-ISO). Compared to the C-ISO control, Intestinal model the CH-ISO diet doubled the production of equol, while with the PR-ISO diet the production of equol in cultures TIM-2 decreased sharply. The CH-ISO diet was also associated with enhanced butyrate production. The numbers of Human faeces fi Intestinal microbiota most bacterial populations analysed did not signi cantly change along cultures with any of the diets. Surprisingly, counts for a gene involved in equol production (tdr) were reduced in all cultures, reflecting a reduction in the number of equol-producing bacteria. In conclusion, under the TIM-2 culture conditions es- tablished, the CH-ISO diet favoured the synthesis of equol.

1. Introduction the modulation of the intestinal microbiota (Graf et al., 2015; Jumpertz et al., 2011; Martinez, Kim, Duffy, Schlegel, & Walter, 2010). Changes Epidemiological data link a soy-rich diet to a reduced risk of de- in the microbial composition of the intestine can lead to associated veloping a number of chronic and degenerative diseases, such as car- metabolic shifts, which in turn may influence equol production. The diovascular and neurodegenerative diseases, osteoporosis and cancer literature contains a host of evidence that diet influences equol pro- (for recent reviews see Zaheer, Humayoun & Akhtar, 2017; Messina, duction too. Poorly digestible carbohydrates have been associated with 2016; Bilal, Chowdhury, Davidson, & Whitehead, 2014). Though soy- increased equol synthesis in fecal cultures, while fructo-oligosacchar- beans contain a collection of biologically-active molecules, isoflavones ides appear to inhibit its production (Decroos, Vanhemmens, Cattoir, may have the strongest influence on human health via their estrogenic Boon, & Verstraete, 2005). The consumption of resistant starch by and antioxidant activities (Messina, 2016). Of all the isoflavone-derived ovariectomized mice under daidzein treatment has been shown to in- metabolites, equol (from daidzein) has the greatest estrogenic (Shor, crease equol formation (Tousen, Abe, Ishida, Uehara, & Ishimi, 2011). Sathyapalan, Atkin, & Thatcher, 2012) and antioxidant activity (Choi & Xylitol too has been shown to enhance equol production from daidzein Kim, 2014). Equol is produced by poorly-characterized members of the in mice (Tamura, Hoshi, & Hori, 2013), and combining daidzein and gut microbiota, most of which belong to the family Coriobacteriaceae lactulose is reported to promote equol production in sows (Zheng, Hou, (Clavel, Lepage, & Charrier, 2014). However, while all the animal Su, & Yao, 2014). In humans it has been reported that vegetarians are species tested so far produce equol in response to soy or daidzein in- more likely to be equol producers than non-vegetarians (59% vs. 25%), take, only 30–60% of humans worldwide seem capable of the same suggesting dietary components other than soy influence equol synthesis (Setchell & Clerici, 2010). Although controversial still exists (Setchell & Cole, 2006). The consumption of milk and dairy products (Pawlowski et al., 2015), some authors speculate that only equol-pro- along with daidzein supplements has also been correlated with en- ducing subjects may fully benefit from soy or isoflavone consumption. hanced equol excretion by equol-producing subjects (Frankenfeld, In vitro and in vivo studies have shown diet to be a pivotal factor in 2011a). Antibiotics, however, have been shown to both increase and

⁎ Corresponding author at: Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain. E-mail address: [email protected] (B. Mayo). https://doi.org/10.1016/j.jff.2020.103819 Received 17 July 2019; Received in revised form 15 January 2020; Accepted 25 January 2020 1756-4646/ © 2020 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

102 CAPÍTULO 3 L. Vázquez, et al. Journal of Functional Foods 66 (2020) 103819 reduce equol production in faecal cultures, and no significant correla- 2.3. Study design tion between any type of antibiotic and equol production has been es- tablished (Atkinson, Berman, Humbert, & Lampe, 2004; Franke et al., Faecal suspensions were thawed in a water bath at 37 °C for 1 h 2012). More data on the nature, extent, and factors affecting equol before being introduced into the system. To standardize the inoculum, synthesis are needed if we are to develop dietary strategies that max- faecal homogenates from the three women were pooled and divided imize endogenous equol production. into four aliquots to independently inoculate each of the four units of Though only human trials can provide definitive proof of the ben- the TIM-2 model (approximately 17.5 mL of the faecal homogenate for efits of equol on human health, animal models (Allred et al., 2005), cell each unit). The units were filled with 120 mL of dialysate solution. cultures (Lehmann, Esch, Wagner, Rohnstock, & Metzler, 2005), arti- Immediately after inoculation, the system was continuously fed with ficial gastrointestinal models (Islam et al., 2014), and faecal fermen- Standard Ileal Efflux Medium (SIEM diet) at a rate of 2.5 mL/h, and the tations (Atkinson et al., 2004) are valuable tools for studying dietary microbiota allowed to adapt for 16 h. SIEM diet simulates the nutritive factors involved in equol synthesis and the microbial and metabolic components characteristic of a common Western diet that reach the interactions involved. The TIM-2 is a validated, computer-controlled, colon (Maathuis et al., 2012); briefly, it contains (g/L) Tween 80 (34.0; dynamic in vitro model of the human colon (Venema, 2015) that has product reference P1365), casein (47.0; C7078), bactopeptone (47.0; been used to assess the effects and interactions of many food compo- 211677), ox bile (0.8; 70168), non-starch polysaccharides [citrus peel nents in the gut (Maathuis, van den Heuvel, Schoterman, & Venema, pectin (9.4; P9135), xylan-(beechwood) (9.4; X4252), arabinogalactan 2012; van Nuenen, Meyer, & Venema, 2003). from larch (9.4; 10830), amylopectin from potato (9.4; A8515)], and The present study uses the TIM-2 gut model to examine equol soluble raw potato starch (78.4; 177130010). All products were pur- production under different dietary regimens. For this, the TIM-2 was chased from Sigma-Aldrich (St. Louis, MO, USA), except bacto peptone inoculated with a homogenized faecal cocktail from three equol-pro- from Thermo Fisher Scientific (Waltham, MA, USA) and potato starch ducing women. The TIM-2 was then fed with a concentrate of iso- from Chem-Supply (Port Adelaide, Australia). SIEM also contained vi- flavones and three distinct diets: (i) a balanced diet (simulating the tamins: (mg/L) menadione (1), D-biotin (2), (0.5), pan- components that reach the colon in a normal diet), (ii) a carbohydrate- tothenate (10), nicotinamide (5), p-aminobenzoic acid (5), and thia- rich diet, (iii) and a protein-rich diet. As a negative control, a balanced mine (4); all from Sigma-Aldrich. The pH was adjusted to 5.8. After diet without isoflavones was also used. adaptation, the system was deprived of SIEM for 2 h so that all available carbohydrates in the system were used up, clearing the way for max- imum microbial utilization of the components in the test diets. Samples 2. Materials and methods were collected after this starvation period (time 0) and the system then fed an experimental diet. 2.1. Stool samples from menopausal women The experimental diets were based on modifications to the SIEM control diet (C diet). For the carbohydrate-rich diet (CH diet), this in- After ethical approval and written informed consent, stool samples volved adding an extra dose of 7.5 g of starch per day. For the protein- were obtained by standardized methods in compliance with the rich diet (PR diet), the SIEM was modified by diluting with TBCO Declaration of Helsinki from three equol-producing menopausal (Tween 80, casein, bactopeptone, and ox bile) 6.25 times and replacing women, whose equol-producer status had been determined in a pre- all carbohydrates with 15 g of casein per day. The C, CH, and PR diets vious study (Guadamuro et al., 2015). Freshly voided stools were col- were then supplemented with one Fisiogen tablet (Zambon, Bresso, lected by the volunteers in plastic containers in which anaerobic con- Italy) per day containing 80 mg of an isoflavone concentrate consisting ditions were maintained using Anaerocult A (Merck, Darmstadt, in > 97% of the glycosides daidzin (60%) and genistin (40%), gen- Germany). Immediately after collection the samples were transported erating the final C-ISO, CH-ISO, and PR-ISO experimental diets. The to the laboratory and pre-processed within an hour in a Mac500 isoflavone content of these diets simulated the daily intake of the anaerobic chamber (Don Whitley Scientific, Bingley, UK) containing an products recommended by the manufacturer. The negative control diet anoxic atmosphere (10% H , 10% CO , and 80% N ). Aliquots of 8.75 g 2 2 2 was SIEM with no isoflavones (C diet). of the faeces were suspended in an equal volume of physiological saline Samples of the TIM-2 lumen content and the dialyzed eluate were dialysate containing 30% glycerol and stored at −80 °C until analysis. taken at, 24, 48 and 72 h. Samples were snap-frozen in liquid nitrogen The components of the dialysate (per litre) were: 2.5 g K HPO ·3H O, 2 4 2 (−196 °C) immediately after collection and stored at −80 °C until 4.5 g NaCl, 0.5 g MgSO , 0.45 g CaCl ·2H O, 0.4 g cysteine-HCl, 0.005 g 4 2 2 analysis. All experiments with all diets were performed in duplicate. FeSO ·7H O, 0.05 g ox-bile, and 1 mL of a vitamin solution (1 mg 4 2 TIM-2 is a computer-controlled model used during the last decades that menadione, 2 mg biotin, 0.5 mg vitamin B , 10 mg pantothenic acid, 12 has been shown to be highly reproducible (Aguirre, Ramiro-Garcia, 5 mg nicotinamide, 5 mg p-aminobenzoic acid, and 4 mg thiamine per Koenen, & Venema, 2014;Rose et al., 2010). Experiments were per- litre). formed in series of two per diet formulation (n = 2).

2.2. TIM-2 experiments 2.4. Analysis of isoflavone metabolites The study was performed in a TIM-2 model simulating the dynamic conditions of the human proximal colon (Venema, 2015). Briefly, this Isoflavone metabolites, including equol, were extracted from 3 mL model consists of four glass compartments with an inner flexible of the lumen content and the dialyzed eluate by solid phase extraction membrane that simulates peristaltic movements and avoids phase se- (SPE) and quantified by ultra-high-performance liquid chromatography paration of solid and liquids in the lumen content. The system is con- (UHPLC), according to Redruello et al. (2015). Briefly, extraction was stantly flushed with nitrogen to maintain anaerobic conditions. In each performed in a Vac Elut-12 Manifold Processing Station (Agilent unit, the volume is maintained at a constant 120 mL and the pH kept Technologies, Santa Clara, CA, USA) with Bond Elut-C18 200 mg SPE above 5.8 by neutralizing the acids produced by the microbiota with cartridges (Agilent Technologies). Samples were mixed with an equal 1 M NaOH. The above membrane also acts as a dialysis system that volume of sodium acetate buffer (0.1 M, pH 4.5), loaded into the solid maintains physiological concentrations of microbial metabolites, such phase cartridge, and washed. After elution, samples were stored in as electrolytes and short-chain fatty acids (SCFAs), preventing the in- opaque vials and kept at −20 °C until analysis. hibition or death of gut microbes that would be caused if the latter were allowed to accumulate.

103 CAPÍTULO 3 L. Vázquez, et al. Journal of Functional Foods 66 (2020) 103819

2.5. DNA extraction numbers of cells of the targeted microbial groups (Supplementary Table 1). The limit of detection was established at around a Ct of 30. Total microbial DNA from the TIM-2 samples was extracted using an The recovery efficiency of DNA extraction from TIM-2 samples was improved protocol for PCR-quality community DNA (Yu & Morrison, assessed by adding (in independent experiments) known numbers of 2004), with some modifications in the lysis step. Briefly, 2 mL of lumen cells from the non-intestinal species Lactococcus garvieae and Serratia samples were centrifuged at 14,000 rpm for 10 min and the cell pellet marcescens. Total microbial DNA from the samples was then extracted, mixed with 200 μL of lysis solution (20 mM Tris- HCl pH 8.0, 2 mM purified and subjected to qPCR analysis as above. EDTA, 1.20% Triton X-100, and 20 mg/mL lysozyme). Twenty units of mutanolysin (Merck, Darmstadt, Germany) and 5 µL of a 2 mg/mL ly- 2.8. Analysis of faecal metabolites sostaphin solution (Sigma-Aldrich) were added to the mixture and in- cubated at 37 °C for 40 min. 1.2 mL of the lysis buffer from the QIAamp SCFA, branched chain fatty acids (BCFAs), lactate and succinate DNA Stool Minikit (Qiagen, Hilden, Germany) were then added, and the were determined by ion chromatography as describe by Cuevas, mixture loaded into a screw-cap tube containing one sea pearl and Alegría, Lagarda, and Venema (2019). In short, both lumen content zirconia/silica beads (0.3 g/0.5 mm and 0.1 g/0.1 mm) (BioSpec Pro- (1.5 mL) and dialyzed eluate (2 mL) samples were centrifuged at ducts, Bartlesville, OK, USA). Mechanical disruption was performed 14,000 rpm for 10 min, filtered through a 0.45 μm PFTE filter, and using a FastPrep FP120 Cell Disrupter (Qbiogene, Carlsbad, CA, USA), diluted with the mobile phase (1.5 mM aqueous sulfuric acid). Meta- employing three cycles at 5.5 m/s for 30 s. Samples were cooled for bolite analysis was performed by ion exclusion chromatography (IEC) 1 min on ice between cycles. Cell extracts were then incubated for using an 883 Basic IC chromatograph (Metrohm, Herisa, Switzerland) 10 min at 95 °C to inactivate enzymes. An inhibitEX tablet (Qiagen) was equipped with a Transgenomic IC Sep ICE-ION-300 column then added to the supernatants, and stool particles and inhibitors bound (30 cm × 7.8 mm × 7 μm) and a MetroSep RP2 Guard. Ten microliters to the tablet’s matrix pelleted by centrifugation. The precipitation of were loaded into the column with the help of an automatic sampler 730 nucleic acids, the removal of RNA and protein, and the purification (Metrohm). The SCFA BCFAs were eluted according to their pKa steps were performed following the protocol of the column supplier (column flow 0.4 mL/min at 65 °C). The acids were detected using (Qiagen). Finally, DNA was eluted with 150 μL of sterile molecular suppressed conductivity detection. The above tests were performed by biology grade water (Sigma-Aldrich) and stored at −20 °C until ana- the Brightlabs company (Venlo, The Netherlands). lysis. 2.9. Statistical analysis 2.6. Real time quantitative (qPCR) A one-way ANOVA and a subsequent Bonferroni post-hoc test were The quantification of the different bacterial groups was performed used to compare absolute quantification of faecal microbial groups and by qPCR using group-specific 16S rDNA sequence-based primers re- SCFAs and BCFAs between sampling times (0, 24, 48, 72 h) in each ported in the literature (Supplementary Table 1). Additionally, the tested diet (C, C-ISO, CH-ISO, PR-ISO), after checking the normality of equol-producing bacteria in the TIM-2 samples were enumerated by the data by the Shapiro-Wilk test. For SCFAs and BCFAs values were qPCR amplification of the tdr gene (the product of which is involved in artificially set to zero before feeding with the different test diets (t = 0). the synthesis of equol) as previously reported (Vázquez, Guadamuro, Statistical analyses were carried out using SPSS software V25.0 (IBM, Giganto, Mayo, & Flórez, 2017). All qPCR reactions were performed in Armonk, NY, USA). Differences were considered to be significant at a p triplicate in 96-well optical plates (Applied Biosystems, Foster City, CA, value of < 0.001 and < 0.05 for the microbial and fatty acids data, USA) using a 7500 Fast Real-Time PCR System (Applied Biosystems), respectively. and in a final volume of 20 µL containing 2 × SYBR Green PCR Master Mix (Applied Biosystems), 0.45 μM of each primer, and 2 μL of template 3. Results DNA (5–10 ng). The standard protocol for the amplification and de- tection was as follows: one cycle for initial denaturation at 95 °C for Table 1 shows the results of the UHPLC analysis of samples of the 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s, and lumen content under the different dietary regimens. Daidzein and di- 1 min at 60 °C for annealing and extension. The cycling conditions for hydrogenistein could not be independently quantified since, under the amplification of the genera Slackia and Eggerthella were slightly mod- chromatographic conditions used, these compounds co-elute (same re- ified: an initial cycle at 95 °C for 10 min, followed by 40 cycles at 90 °C tention time). for 15 s, and 1 min at 62 °C (Cho et al., 2016). Melting curve analysis Analysis of samples from the dialyzed eluates showed that none of (Tm) was performed after all amplifications to confirm the specificity of the isoflavones or their metabolites crossed the dialysis membrane the qPCR reaction. Results were expressed in absolute quantities using (data not shown). As expected, when the TIM-2 was inoculated with the logarithmic units of colony forming units per millilitre (see below). control (C) diet, neither isoflavones nor their derived metabolites were ever detected. Variable concentrations of daidzein (plus dihy- 2.7. Standard curves, limit of detection and recovery efficiency drogenistein), genistein, dihydrodaidzein, O-DMA and equol were ob- tained at the different sampling points with all the isoflavone-con- The absolute quantification of different microbial groups was per- taining diets under analysis. In general, O-DMA and equol increased formed using standard curves of representative species for each group over time with a maximum content reached at 72 h. Compared to the C- grown under appropriate conditions (Supplementary Table 2). For mi- ISO diet, the CH-ISO diet doubled the production of equol at 48 (5.67 v. crobial enumeration, 100 µL of overnight cultures were used to prepare 11.22 μM) and 72 h (8.59 v. 18.31 μM). In contrast, both equol and O- 10-fold serial dilutions for determining viable counts by plating in DMA synthesis decreased sharply with the PR-ISO diet (approx. three duplicate. From the same cultures, total genomic DNA was isolated times that seen with the C-ISO diet). Equol production was not detected from 1 mL of the cultures using the GenElute Bacterial Genomic DNA (limit of quantification 3.2 μM) when the TIM-2 model was inoculated Kit (Sigma-Aldrich) following the manufacturer’s instructions. Standard with faecal homogenates from an equol non-producing woman (data curves were generated with 10-fold serial dilutions of the purified DNA not shown). as a template in qPCR reactions with the corresponding group-specific The limit of detection of the qPCR analyses ranged from 0.5 × 102 4 primers. Primer efficiency for each primer pair was calculated from the cfu/mL (1.70 Log10 cfu/mL) (Atopobium) to 1.75 × 10 cfu/mL (4.24 − slope of the standard curve (E = 10 1/slope). The limit of detection of Log10 cfu/mL) (Enterobacteriaceae). Similar recovery efficiencies were the qPCR analyses was calculated by diluting purified DNA from known obtained by adding known numbers of cells of the Gram-positive

104 CAPÍTULO 3 L. Vázquez, et al. Journal of Functional Foods 66 (2020) 103819

Table 1 Effect of different diets on the concentration of equol and other isoflavones metabolites in TIM-2 samples inoculated with a faecal homogenate from three equol- producing women.

Dieta Sampling time (h) Isoflavones metabolites (µM ± SD)

Daidzein + dihydro-genisteinb Genistein Dihydro-daidzein O-DMA Equol

C0 −−−−− C24 −−−−− C48 −−−−− C72 −−−−−

C–ISOc 0 −−3.54 ± 2.68 0.33 ± 0.40 − C–ISO 24 14.92 ± 10.55 0.67 ± 0.46 8.32 ± 3.03 78.65d 1.25 ± 0.41 C–ISO 48 52.23 ± 36.90 9.87 ± 12.77 14.25 ± 0.63 89.96 ± 54.70 5.67 ± 0.19 C–ISO 72 71.04 ± 50.20 1.92 20.83 ± 2.58 95.48 ± 78.64 8.59 ± 1.71

CH–ISOc 0 −−3.92 ± 2.74 0.39 ± 0.25 − CH–ISO 24 > 200 μM 6.72 ± 3.42 7.30 ± 6.12 39.86 ± 15.41 1.65 ± 2.05 CH–ISO 48 197.01 ± 139.2 22.40 10.83 ± 8.71 66.41 ± 16.50 11.22 ± 15.16 CH–ISO 72 123.45 ± 87.23 7.39 9.22 ± 2.60 131.9 ± 71.14 18.31 ± 21.87

PR–ISOc 0 −−5.06 ± 4.36 0.13 ± 0.05 − PR–ISO 24 > 200 μM > 200 μM > 200 μM 0.22 ± 0.13 0.55 ± 0.62 PR–ISO 48 > 200 μM > 200 μM > 200 μM 2.38 ± 0.34 1.66 ± 0.51 PR–ISO 72 > 200 μM > 200 μM > 200 μM 7.62 ± 1.78 2.71 ± 1.66

−, not detected. a Diets: C, control diet (Standard Ileal Efflux Media, SIEM); C-ISO, control diet with isoflavones (SIEM supplemented with soy extract); CH-ISO, carbohydrate-rich diet (SIEM supplemented with starch and isoflavones); PR-ISO, protein-rich diet (SIEM supplemented with soy extract and isoflavones). b Daidzein and dihydrogenistein are co-eluents under the UHPLC conditions established in this work. c The isoflavone supplement was shown to be composed of > 96.76% of isoflavone glycosides, daidzin (59.73%) and genistin (40.27%). d Analyses having no SD values had no replicates.

Lactococcus garvieae and the Gram-negative Serratia marcescens (89.8 4. Discussion and 87.4%, respectively) to the TIM-2 samples (Supplementary Fig. 1). Thus, the real counts for the targeted microbial groups should be a little It is well established that the frequency of equol producers among higher than those reported in Table 2. In terms of absolute numbers, no Western adults ranges from 25 to 35% (Setchell & Cole, 2006; Peeters major changes were observed in the dominant populations throughout et al., 2007). The amount of equol actually produced varies widely the incubation period with the C-ISO diet, except for that of Lactoba- among subjects, even after the consumption of an identical quantity of cillus spp., which increased significantly with all isoflavone-containing isoflavones (Setchell & Cole, 2006; Franke et al., 2012; Setchell et al., diets and the C diet (Table 2; Supplementary Fig. 2). Reduced numbers 2013; Guadamuro et al., 2015). Understanding why equol production of Bacteroidetes were also observed with the CH-ISO diet. Numbers for varies so much is important since the health benefits of isoflavones the Coriobacteriaceae, Atopobium spp. and Slackia spp. were similar appear to be greater in equol producers (Yoshikata, Myint, Ohta, & with all isoflavone-containing diets, and remained constant throughout Ishigaki, 2019; Tousen et al., 2011). Beyond the composition and ac- the sampling time points. In contrast, Eggerthella numbers followed a tivity of the individual gut microbiota – a key factor in equol formation reducing trend with all the diets tested except for PR-ISO (particularly (Clavel et al., 2014; Setchell and Clerici, 2010), it is important to de- at 24 and 48 h). A significant reduction in numbers was clearly seen via termine what conditions enhance equol production (Brown et al., 2014; the tdr gene count (Table 2; Supplementary Fig. 2). Setchell et al., 2013). A lack of SCFA synthesis was observed after the initial starvation In the present work, the artificial colon was inoculated with the period; this was interpreted as reflecting the depletion of all fermen- same pooled faecal homogenate from three equol-producing women table carbohydrates. A linear cumulative production of SCFAs and (Guadamuro et al., 2015). The use of inocula of pooled faecal samples BCFAs was observed for all isoflavone-containing diets over the 72 h of reduces the high intra-individual variation in the composition and ac- TIM-2 incubation (see Supplementary Fig. 3 for an example [C-ISO tivity of the microbiota recorded when cultures are inoculated with fermentation]). The main fatty acids produced by the microbiota of the faeces from a single donor (Aguirre et al., 2014). However, large range pooled homogenate were acetate, propionate and butyrate (Table 3; of the results for the study variables in different replicates of the present Supplementary Fig. 4). The presence of isoflavones in the culture tests was noted. This might be attributed to some heterogeneity in each medium did not significantly influence the production of any of the of the faecal homogenates, which were independently produced. fatty acids analyzed. At the 72 h sampling point, the production of total As expected, equol was not detected when isoflavones were absent. volatile fatty acids in the control diets with (C-ISO, 119.80 mmol) and The same negative results were also obtained when the TIM-2 was in- without (C, 120.6 mmol) isoflavones was similar, while production oculated with faeces from an equol non-producing woman (data not increased with both the CH-ISO (160.28 mmol) and PR-ISO shown). Compared to the C-ISO diet, equol production was greatly (142.02 mmol) diets. The production of acetate did not significantly enhanced with the CH-ISO medium. These results are in agreement with change under any of the dietary regimens. In contrast, the CH-ISO diet those reported in the literature which indicate an increase in equol greatly enhanced the production of butyrate while reducing the pro- formation in faecal cultures in the presence of poorly digestible car- duction of propionate (Table 3). The PR-ISO diet notably increased the bohydrates (Decroos et al., 2005), and in model animals fed resistant production of propionate, iso-butyrate, iso-valerate and other fatty acids starch (Tousen et al., 2016; Tousen et al., 2011), xylitol (Tamura et al., (caproic acid, formic acid, lactic acid, succinic acid, and valeric acid), 2013) or lactulose (Zheng et al., 2014). Compared to the C-ISO diet, while reducing the production of butyrate. equol production fell by some two thirds at both 48 and 72 h with the PR-ISO diet. Further, the same diet also seemed to inhibit the formation of O-DMA, a non-estrogenic daidzein metabolite (with the CH-ISO diet

105 .Vzuz tal. et Vázquez, L.

Table 2 Absolute quantification of faecal microbial populations and tdr genes analysed by qPCR during TIM-2 fermentations inoculated with a faecal homogenate from three equol-producing women.

a c Diet Sampling Microbial group/gene (Log10 cfu/mL ± SD) timeb Bacteroidetes Clostridium leptum Clostridium Bifidobacterium spp. Enterobacteriaceae Lactobacillus spp. Coriobacteriaceae Atopobium cluster Eggerthella spp. Slackia spp. tdr genec phylum group coccoides group

C 0 10.05 ± 0.04 7.92 ± 0.04 7.72 ± 0.07 7.63 ± 0.12 8.00 ± 0.10 4.54 ± 0.29 8.42 ± 0.09 7.65 ± 0.09 5.02 ± 0.18 4.24 ± 0.18 3.47 ± 0.32 C 24 10.21 ± 0.05 8.08 ± 0.05 7.82 ± 0.14 7.48 ± 0.16 9.01 ± 0.18* 5.47 ± 0.13 8.30 ± 0.10 7.55 ± 0.06 4.81 ± 0.23 4.21 ± 0.17 3.15 ± 0.13 C 48 10.07 ± 0.05 7.98 ± 0.06 7.81 ± 0.03 7.44 ± 0.10 8.88 ± 0.12* 6.13 ± 0.11* 8.19 ± 0.11 7.32 ± 0.09 4.90 ± 0.12 4.41 ± 0.10 3.06 ± 0.26 C 72 9.90 ± 0.02 7.89 ± 0.08 7.67 ± 0.11 7.45 ± 0.09 8.90 ± 0.13* 6.73 ± 0.09* 8.08 ± 0.07 7.34 ± 0.06 5.10 ± 0.02 4.81 ± 0.08* 3.07 ± 0.21

C-ISO 0 9.92 ± 0.07 7.93 ± 0.09 7.76 ± 0.03 7.46 ± 0.10 7.74 ± 0.10 5.05 ± 0.06 8.45 ± 0.07 7.60 ± 0.08 5.11 ± 0.15 4.22 ± 0.16 3.58 ± 0.53 C-ISO 24 9.93 ± 0.08 7.61 ± 0.13 7.31 ± 0.05 7.24 ± 0.17 8.27 ± 0.28 6.34 ± 0.13* 8.04 ± 0.09* 7.14 ± 0.11* 4.72 ± 0.09* 4.19 ± 0.18 3.21 ± 0.42 C-ISO 48 9.92 ± 0.06 7.75 ± 0.10 7.46 ± 0.14 7.73 ± 0.14 8.70 ± 0.07 7.03 ± 0.09* 8.20 ± 0.04 7.51 ± 0.05 4.89 ± 0.02 4.32 ± 0.11 2.84 ± 0.97 106 C-ISO 72 9.93 ± 0.12 7.82 ± 0.09 7.61 ± 0.08 7.55 ± 0.12 8.69 ± 0.12 6.92 ± 0.09* 7.99 ± 0.06* 7.31 ± 0.05 4.87 ± 0.12 4.62 ± 0.08 3.17 ± 0.20

CH-ISO 0 10.04 ± 0.05 7.93 ± 0.10 7.86 ± 0.03 7.49 ± 0.12 8.04 ± 0.12 4.74 ± 0.04 8.37 ± 0.05 7.61 ± 0.04 4.98 ± 0.14 4.25 ± 0.15 3.61 ± 0.40 CH-ISO 24 9.73 ± 0.07 8.04 ± 0.04 8.04 ± 0.14 7.26 ± 0.15 8.66 ± 0.13 5.65 ± 0.07* 8.20 ± 0.06 7.45 ± 0.05 4.64 ± 0.13 4.20 ± 0.17 2.95 ± 0.37 CH-ISO 48 9.16 ± 0.03* 7.76 ± 0.04 8.00 ± 0.11 7.38 ± 0.18 8.78 ± 0.18 6.11 ± 0.06* 8.40 ± 0.04 7.54 ± 0.06 4.53 ± 0.13* 4.22 ± 0.18 2.80 ± 0.20 CH-ISO 72 8.72 ± 0.08* 7.31 ± 0.10* 7.88 ± 0.08 8.65 ± 0.17* 8.68 ± 0.14 6.65 ± 0.07* 9.00 ± 0.03* 8.22 ± 0.08* 4.43 ± 0.13* 4.64 ± 0.16 2.58 ± 0.14

PR-ISO 0 9.83 ± 0.07 7.82 ± 0.10 7.52 ± 0.16 7.33 ± 0.14 8.35 ± 0.21 5.16 ± 0.12 8.07 ± 0.05 7.33 ± 0.09 4.74 ± 0.12 4.21 ± 0.17 3.39 ± 0.39 PR-ISO 24 9.87 ± 0.04 7.49 ± 0.08 7.47 ± 0.06 7.52 ± 0.16 8.38 ± 0.14 6.28 ± 0.11* 8.18 ± 0.08 7.31 ± 0.12 4.99 ± 0.10 4.24 ± 0.17 3.28 ± 0.23 PR-ISO 48 9.99 ± 0.07 7.39 ± 0.08 7.48 ± 0.07 7.55 ± 0.23 8.36 ± 0.28 7.01 ± 0.09* 8.16 ± 0.10 7.43 ± 0.10 4.95 ± 0.09 4.34 ± 0.23 3.15 ± 0.27 PR-ISO 72 9.79 ± 0.06 7.00 ± 0.10* 7.37 ± 0.14 7.36 ± 0.21 8.10 ± 0.32 7.01 ± 0.09* 7.89 ± 0.13 7.43 ± 0.08 4.79 ± 0.14 4.60 ± 0.29 2.97 ± 0.43

The asterisk (*) indicates statistical differences as compared to t = 0 each (p value < 0.001). a Diets: C, control diet (Standard Ileal Efflux Media, SIEM); C-ISO, control diet with isoflavones (SIEM supplemented with soy extract); CH-ISO, carbohydrate-rich diet (SIEM supplemented with starch and isoflavones); PR-ISO, protein-rich diet (SIEM supplemented with soy extract and isoflavones). b TIM-2 cultures were sampled at 0 (before adding the diets of the study), 24, 48 and 72 h. c The limit of detection ranged from 1.70 Log10 cfu/mL (Atopobium) to 4.24 Log10 cfu/mL (Enterobacteriaceae). Journal ofFunctionalFoods66(2020)103819 CAPÍTULO 3 CAPÍTULO 3 L. Vázquez, et al. Journal of Functional Foods 66 (2020) 103819

Table 3 Average cumulative production of SCFAs and BCFAs during TIM-2 fermentation inoculated with faecal samples from equol-producing women.

Dieta Sampling time (h) Fatty acid (mmol)b

Acetate Butyrate Propionate Iso-butyrate Iso-valerate Othersc

C0 0 0 0 0 0 0 C 24 14.00 ± 4.88 18.73 ± 1.99 4.25 ± 2.08 0.28 ± 0.35 0.57 ± 0.24 1.11 ± 0.36 C 48 28.65 ± 8.42 42.44 ± 0.26 8.58 ± 2.31 0.58 ± 0.65 1.47 ± 0.45 2.27 ± 0.60 C 72 40.88 ± 10.22 61.59 ± 1.20 11.86 ± 2.70 1.00 ± 0.70 2.21 ± 0.23 3.08 ± 0.80

C-ISO 0 0 0 0 0 0 0 C-ISO 24 14.75 ± 0.53 16.74 ± 3.41 5.61 ± 0.64 0.59 ± 0.33 0.80 ± 0.37 0.75 ± 0.43 C-ISO 48 30.27 ± 0.94 36.87 ± 8.82 10.79 ± 0.30 1.36 ± 0.41 1.96 ± 0.38 2.51 ± 0.63 C-ISO 72 42.40 ± 3.49 55.05 ± 7.58 14.95 ± 1.19 1.89 ± 0.71 2.78 ± 0.80 2.73 ± 0.69

CH-ISO 0 0 0 0 0 0 0 CH-ISO 24 12.71 ± 3.70 31.78 ± 1.87*† 3.43 ± 1.02 0.15 ± 0.20 0.33 ± 0.52 3.55 ± 0.82 CH-ISO 48 19.63 ± 6.47 66.12 ± 4.00† 5.97 ± 2.42 0.25 ± 0.37 0.64 ± 0.94 7.56 ± 1.41 CH-ISO 72 38.58 ± 22.56 93.07 ± 5.03*† 8.47 ± 2.99 0.32 ± 0.49 0.84 ± 1.28 19.00 ± 2.39

PR-ISO 0 0 0 0 0 0 0 PR-ISO 24 11.63 ± 0.05 11.66 ± 0.42 6.68 ± 0.69 1.62 ± 0.64 3.49 ± 0.85*† 1.33 ± 0.53 PR-ISO 48 28.40 ± 1.31 29.92 ± 1.73 16.46 ± 1.16 5.32 ± 1.05*† 10.79 ± 1.32*† 4.82 ± 1.57 PR-ISO 72 45.01 ± 1.81 45.11 ± 0.98*† 24.62 ± 0.63* 8.51 ± 0.99*† 17.19 ± 1.32*† 7.58 ± 2.35

The asterisk (*) and the cross (†) indicate statistically significant differences at a specific time point compared, respectively, to the control diet (C) and to the control diet with isoflavones(C-ISO) (p < 0.05). a Diets: C, control diet (Standard Ileal Efflux Media, SIEM); C-ISO, control diet with isoflavones (SIEM supplemented with soy extract); CH-ISO, carbohydrate-rich diet (SIEM supplemented with starch and isoflavones); PR-ISO, protein-rich diet (SIEM supplemented with proteins and isoflavones). b Average results of three independent analyses ± SD are shown. c Other volatile fatty acids: caproic acid, formic acid, lactic acid, succinic acid, and valeric acid. its synthesis was not clearly affected). Despite being non-estrogenic, O- which suggests there might be coriobacteria species or strains that do DMA might also be important with respect to the risk of developing not produce equol. An in vivo human trial has recently reported that some diseases, or in their progression (Frankenfeld, 2011b). Although counts for tdr and related functional genes did not increase significantly no significant differences in total protein intake have been observed during isoflavone consumption (Vázquez et al., 2017). Even more sur- between equol producers and non-producers (Yoshikata et al., 2019; prisingly, in the present work, the number of tdr genes decreased sig- Setchell et al., 2013), the PR-ISO diet had a strongly negative effect on nificantly over incubation time. equol production in the present TIM-2 system. As equol is a bacterial metabolite produced mostly by intestinal In general, except in punctual sampling points, the tracked bacterial species of the family Coriobacteriaceae (Clavel et al., 2014), the present populations did not significantly change during the 72 h of culture with data suggest that certain dietary carbohydrates promote somehow the any of the diets assayed, excluding that of Enterobacteriaceae and equol-producing activity, but not the equol-producing bacterial num- Lactobacillus spp. counts. These became significantly larger with almost bers. In contrast, the presence of high protein content inhibits in some all four diets tested, which suggests unspecific isoflavone-independent way the synthesis of this compound. The many bacterial interactions increases. These counting results further suggest that the tested taking place in the intestines (Browne et al., 2016), and in the faecal (dominant) faecal populations are a priori not involved in isoflavone cultures of this study (cross feeding, activation and repression of gene metabolism, or influenced by the presence of these compounds. Given expression, increase supply of precursors, etc.), impedes at the moment their high β-glucosidase activity, Lactobacillus spp., might participate in to anticipate a hypothesis on the basis for the equol promotion or in- the release of aglycones from isoflavone glycosides but they are not hibition observed in this study. involved in the subsequent transformation of these compounds The different diets were associated with statistically significant (Delgado, Guadamuro, Flórez, Vázquez, & Mayo, 2019; Landete et al., changes in the cumulative production of SCFAs and BCFAs. The total 2016). Similarly, the numbers of Coriobacteriaceae and specific groups amount and the main specific fatty acids produced varied widely across within this family, of which some are known to be involved in equol the tested diets. Compared to the C-ISO diet, the production of butyrate production (Clavel et al., 2014; Harmsen et al., 2000), did not sig- increased by about 50% with the CH-ISO diet, while the production of nificantly change over fermentation with any tested diet. Thus, the this fatty acid was reduced by 25% with the PR-ISO diet. production of equol was not associated with the presence of, or the Simultaneously, the production of propionate (50%), iso-butyrate (8- increase in, any of the bacterial populations examined. Equol-producing fold) and iso-valerate (8-fold) increased with the PR-ISO diet. These bacteria may be present in such small numbers (see below) that their results are not surprising since carbohydrates and proteins are the main detection and quantification is precluded even by state-of-the-art high- source of SCFAs and BCFAs, respectively (Ríos-Covián et al., 2016; throughput DNA sequencing technologies, as reported in recent pro- Flint, Duncan, Scott, & Louis, 2015). Acetate, propionate and butyrate spective studies (Guadamuro, Azcárate-Peril, Tojo, Mayo, & Delgado, are key microbial metabolites in the physiology (source of energy, 2019; Guadamuro, Dohrmann, Tebbe, Mayo, & Delgado, 2017). modulator of biosynthesis of blood lipids and insulin secretion) and The absolute quantification of tdr, which encodes a tetra- health (inhibition of pathogens, anti-proliferative and anti-in- hydrodaidzein reductase essential for equol production (Schröder, flammatory properties) of the human gastrointestinal tract (Morrison & Matthies, Engst, Blaut, & Braune, 2013; Tsuji, Moriyama, Nomoto, & Preston, 2016; Ríos-Covián et al., 2016). The presence of butyrate in

Akaza, 2012), was around 3.5 Log10 units per mL of sample in the large quantities has been associated with a reduction in the risk of in- different cultures, suggesting that equol-producing bacteria were not testinal diseases (Pozuelo et al., 2015; Machiels et al., 2014), and selected for by any of the tested culture conditions. This amount re- polysaccharides, especially resistant starches, have been reported to presents about one logarithmic unit lower than the smallest population promote butyrate synthesis by the gut microbiota (Ho, Kosik, of equol-producing bacteria quantified by qPCR (that of Slackia spp.), Lovegrove, Charalampopoulos, & Rastall, 2018; Shen et al., 2017).

107 CAPÍTULO 3 L. Vázquez, et al. Journal of Functional Foods 66 (2020) 103819

However, neither the amount of butyrate, nor of other SCFAs, appeared Chemistry, 53, 8542–8550. to be associated with the presence of isoflavones in the present culture Atkinson, C., Berman, S., Humbert, O., & Lampe, J. W. (2004). In vitro incubation of human feces with daidzein and antibiotics suggests interindividual differences in the system. bacteria responsible for equol production. Journal of Nutrition, 134, 596–599. Bilal, I., Chowdhury, A., Davidson, J., & Whitehead, S. (2014). Phytoestrogens and pre- 5. Conclusions vention of breast cancer: The contentious debate. World Journal of Clinical Oncology, 5, 705–712. Brown, N. M., Galandi, S. L., Summer, S. S., Zhao, X., Heubi, J. E., King, E. C., & Setchell, The present results show that equol synthesis requires the presence K. D. (2014). S-(-)equol production is developmentally regulated and related to early of isoflavones in the diet. Equol only appeared in TIM-2 cultures in- diet composition. Nutrition Research, 34, 401–409. oculated with faeces from equol-producing women. The amount of Browne, H. P., Forster, S. C., Anonye, B. O., Kumar, N., Neville, B. A., Stares, M. D., ... Lawleym, T. D. (2016). Culturing of 'unculturable' human microbiota reveals novel equol produced increased over incubation time, with the highest con- taxa and extensive sporulation. Nature, 533, 543–546. centration obtained in all cases at 72 h. At this point, the amount of Cho, G. S., Ritzmann, F., Eckstein, M., Huch, M., Briviba, K., Behsnilian, D., ... Franz, C. M. fi equol produced with the CH-ISO diet was double to that obtained with A. P. (2016). Quanti cation of Slackia and Eggerthella spp. in human feces and ad- hesion of representatives strains to Caco-2 cells. Frontiers in Microbiology, 7,1–10. the C-ISO diet. In contrast, the amount of equol produced with the PR- Choi, E. J., & Kim, G. H. (2014). The antioxidant activity of daidzein metabolites, O- ISO diet was about half that of the same control. A carbohydrate-rich desmethylangolensin and equol, in HepG2 cells. Molecular Medicine Reports, 9, – diet therefore seems to favour equol production in equol producers. 328 332. Clavel, T., Lepage, P., & Charrier, C. (2014). The family Coriobacteriaceae. In E. Further, in the CH-ISO cultures, butyric acid was enhanced at the ex- Rosenberg, E. F. DeLong, S. Lory, E. Stackebrandt, & F. Thompson (Eds.). The pro- pense of acetic acid. Together these results suggest that a carbohydrate- karyotes-actinobacteria (pp. 201–238). Berlin: Springer-Verlag. rich diet may favour gut health by promoting the production of equol, Cuevas, M., Alegría, A., Lagarda, M. J., & Venema, K. (2019). Impact of plant sterols fl enrichment dose on gut microbiota from lean and obese subjects using TIM-2 in vitro when iso avones are available, or by increasing the butyric acid con- fermentation model. Journal of Functional Foods, 54, 164–174. centration. The in vitro results of this study should be tested in vivo Decroos, K., Vanhemmens, S., Cattoir, S., Boon, N., & Verstraete, W. (2005). Isolation and through experimental human trials, as the equol production response characterisation of an equol-producing mixed microbial culture from a human faecal sample and its activity under gastrointestinal conditions. Archives of Microbiology, may be modulated by the large interindividual heterogeneity of the gut 183,45–55. microbiota. Therefore, further research will be required to unravel the Delgado, S., Guadamuro, L., Flórez, A. B., Vázquez, L., & Mayo, B. (2019). Fermentation links between intestinal microbial populations, carbohydrate-rich diets of commercial soy beverages with lactobacilli and bifidobacteria strains featuring β and enhanced equol production. high -galactosidase activity. Innovative Food Science and Emerging Technologies, 51, 148–155. Flint, H. J., Duncan, S. H., Scott, K. P., & Louis, P. (2015). Links between diet, gut mi- 6. Ethics statement crobiota composition and gut metabolism. Proceedings of the Nutrition Society, 74, 13–22. Franke, A. A., Lai, J. F., Halm, B. M., Pagano, I., Kono, N., Mack, W. J., & Hodis, H. N. Our research did not include any human subjects and animal ex- (2012). Equol production changes over time in postmenopausal women. Journal of periments. Nutritional Biochemistry, 23, 573–579. Frankenfeld, C. L. (2011a). Dairy consumption is a significant correlate of urinary equol concentration in a representative sample of US adults. American Journal of Clinical Author contribution Nutrition, 93, 1109–1116. Frankenfeld, C. L. (2011b). O-Desmethylangolensin: The importance of equol’s lesser LV, SV, and JV contributed to methodology and investigation. ABF known cousin to human health. Advances in Nutrition, 2, 317–324. Graf, D., Di Cagno, R., Fåk, F., Flint, H. J., Nyman, M., Saarela, M., & Watzl, B. (2015). contributed to data curation and formal analysis. KV and BM con- Contribution of diet to the composition of the human gut microbiota. Microbial tributed to conceptualization, funding acquisition and formal analysis. Ecology and Health Diseases, 26, 26164. ABF and BM wrote the original draft. All authors participate in the Guadamuro, L., Delgado, S., Redruello, B., Flórez, A. B., Suárez, A., Martínez-Camblor, P., fi & Mayo, B. (2015). Equol status and changes in fecal microbiota in menopausal review and editing of the nal version of the manuscript. women receiving long-term treatment for menopause symptoms with a soy-iso- flavone concentrate. Frontiers in Microbiology, 6, 777. Declaration of Competing Interest Guadamuro, L., Dohrmann, A. B., Tebbe, C. C., Mayo, B., & Delgado, S. (2017). Bacterial communities and metabolic activity of faecal cultures from equol producer and non- producer menopausal women under treatment with soy isoflavones. BMC The authors declare that they do not have any conflict of interest. Microbiology, 17,93. Guadamuro, L., Azcárate-Peril, M. A., Tojo, R., Mayo, B., & Delgado, S. (2019). Changes in Acknowledgements the faecal microbiota of an equol-producing menopausal woman over six months of dietary supplementation with isoflavone. AIMS Microbiology, 5, 102–116. Harmsen, H. J. M., Wildeboer-Veloo, A. C. M., Grijpstra, J., Knol, J., Degener, J. E., & This study was partly supported by projects from the Spanish Welling, G. W. (2000). Development of 16S rRNA-based probes for the Coriobacterium Ministry of Economy and Competitiveness (MINECO) (AGL-2014- group and the Atopobium cluster and their application for enumeration of Coriobacteriaceae in human feces from volunteers of different age groups. Applied 57820-R) and Asturias Principality (IDI/2018/000114). LV was sup- and Environmental Microbiology, 66, 4523–4527. ported by a contract from MINECO within the FPI Program (BES-2015- Ho, A. L., Kosik, O., Lovegrove, A., Charalampopoulos, D., & Rastall, R. A. (2018). In vitro 072285). In addition, the study was also funded by the Centre for fermentability of xylo-oligosaccharide and xylo-poly-saccharice fractions with dif- ferent molecular weights by human faecal bacteria. Carbohydrate Polymers, 179, Healthy Eating & Food Innovation (HEFI) of Maastricht University, 50–58. Campus Venlo. This research has been made possible with the support Islam, M. A., Punt, A., Spenkelink, B., Murk, A. J., van Leeuwen, R. F. X., & Rietjens, I. M. of the Dutch Province of Limburg. (2014). Conversion of major soy isoflavone glucosides and aglycones in in vitro in- testinal models. Molecular Nutrition and Food Research, 58, 503–515. Jumpertz, R., Le, D. S., Turnbaugh, P. J., Trinidad, C., Bogardus, C., Gordon, J. I., & Appendix A. Supplementary material Krakoff, J. (2011). Energy-balance studies reveal associations between gut microbes, caloric load, and nutrient absorption in humans. 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(-)-equol in healthy adults. Journal of Nutrition, 143, 1950–1958.

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A B 10 Lactococcus garvieae IPLA 31405 10 Serratia marcescens SE41

8 8

6 6

4 4 Log (cfu/mL) Log (cfu/mL) y = -0,283x + 10,764 y = -0,2791x + 10,105 2 R² = 0,9983 2 R² = 0,9998 0 0 0 10 20 30 40 0 10 20 30 40 Ct Ct C 10

8

6 Expected Observed 4 Log (cfu/mL) 2

0 L. garviae S. marcescens Supplementary Figure 1.- Standard curves and limit of detection of Lactococcus lactis (A) and Serratia marcescens (B) by qPCR. 2 Linear regression was obtained plotting the cycle threshold (Ct) values vs. the Log10 of the counting (in cfu/ml). The equation and R value of the regression lines are also indicated. In Panel C, recovery efficiency using serial dilutions of DNA from known amounts of cells added to the TIM-2 samples. 110 CAPÍTULO 3

60%

40%

20%

0% difference %

-20%

-40%

-60% 24 C 24 C 72 C 48 C 24 C 72 C 48 C 24 C 72 C 48 C 24 C 72 C 48 C 24 C 72 C 48 24 C-SE 24 C-SE 72 C-SE 48 C-SE 24 C-SE 72 C-SE 48 C-SE 24 C-SE 72 C-SE 48 C-SE 24 C-SE 72 C-SE 48 C-SE 24 C-SE 72 C-SE 48 24 PR-SE 24 PR-SE 72 PR-SE 48 PR-SE 24 PR-SE 72 PR-SE 48 PR-SE 24 PR-SE 72 PR-SE 48 PR-SE 24 PR-SE 72 PR-SE 48 PR-SE 24 PR-SE 72 PR-SE 48 24 CH-SE 24 CH-SE 72 CH-SE 48 CH-SE 24 CH-SE 72 CH-SE 48 CH-SE 24 CH-SE 72 CH-SE 48 CH-SE 24 CH-SE 72 CH-SE 48 CH-SE 24 CH-SE 72 CH-SE 48

Diet/ sampling point

Bacteroidetes phylum Clostridium coccoides group Enterobacteriaceae Atopobium cluster Slackia spp. Clostridium leptum group Bifidobacterium spp. Lactobacillus spp. Eggerthella spp. Coriobacteriaceae family .

Supplementary Figure 2.- Absolute quantification of faecal microbial populations by qPCR during TIM-2 fermentations inoculated with a faecal homogenate from three equol-producing women. 111 CAPÍTULO 3

70 60

50

) acetate 40 propionate butyrate nmol ( 30 iso butyrate 20 iso valerate Other SCFAs SCFAs and BCFAs and BCFAs SCFAs 10 0 0 24 Time (h) 48 72

Supplementary Figure 3.- Linearity of cumulative SCFAs and BCFAs production during the TIM-2 control fermentation without isoflavones inoculated with a faecal homogenate from three equol-producing women.

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120

100 )

mmol Acetic acid 80 Propionic acid Butyric acid

SCFAs ( SCFAs Others 60

40 Cummulative

20

0 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 C C-ISO CH-ISO PR-ISO Sampling time/Diet

Supplementary Figure 4.- Average cumulative production of SCFAs and BCFAs during TIM-2 fermentation inoculated with faecal samples from three equol-producing women.

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Supplementary Table 1.- Targeted bacterial groups, oligonucleotide primers, efficiency and annealing temperatures used in this study.

a Annealing Target Primer Primer sequence 5´-3´ Efficiency Reference (ºC)

Bact934F GGARCATGTGGTTTAATTCGATGAT Bacteroidetes phylum 1.93 60 Guo et al. (2008) Bact1060R AGCTGACGACAACCATGCAG g-Clept-F GCACAAGCAGTGGAGT Clostridium leptum group 1.84 60 Matsuki et al. (2004) g-Clept-R CTTCCTCCGTTTTGTCAA g-Ccoc-F AAATGACGGTACCTGACTAA Clostridium coccoides group 1.72 60 Matsuki et al. (2002) g-Ccoc-R CTTTGAGTTTCATTCTTGCGAA F-bifido CGCGTCYGGTGTGAAAG Delroisse et al. Bifidobacterium spp. 1.95 60 R-bifido CCCCACATCCAGCATCCA (2008) En-lsu3F TGCCGTAACTTCGGGAGAAGGCA Matsuda et al. Enterobacteriaceae 1.73 60 En-lsu3-R TCAAGGCTCAATGTTCAGTGTC (2007) Lacto-F AGCAGTAGGGAATCTTCCA Lactobacillus spp. 1.80 60 Heilig et al. (2002) Lacto-R CACCGCTACACATGGAG ATO291 GGGTTGAGAGACCGACC Harmsen et al. Coriobacteriaceae 1.57 60 COR653 CCCTCCC(A/C)TACCG GACCC (2000) c-Atopo-F GGGTTGAGAGACCGACC Atopobium cluster 1.92 60 Matsuki et al. (2004) c-Atopo-R CGGRGCTTCTTCTGCAGG Eggfw TACTCCTCGCCCCCCTCCTGG Eggerthella spp. 1.81 62 Cho et al. (2016) Eggrev CTTCTTCTGCAGGTACCGTC Coriofw GACGGTACCTGCAGAAGAAG Slackia spp. 1.96 62 Cho et al. (2016) Slackiarev CCCCGGCTTCGACGGTGCCGCTT

tdr.qPCR-F RTYAACGGCRAYATGCAGGT Vázquez et al. tdr gene 1.99 60 tdr.qPCR-R GGMAYYTCCATGTTGTAGGA (2017)

ITSLg30F ACTTTATTCAGTTT TGAGGGGTCT Lactococcus garvieae 1.92 60 Dang et al. (2012) ITSLg319R TTTAAAAGAATTCGCAGCTTTACA GGTGAGCTTAATACGTTCATCAATT SMSF Serratia marcescens G 1.90 60 Iwaya et al. (2005) SMSR GCAGTTCCCAGGTTGAGCC

aPrimer efficiency was calculated from the slope of the standard curve for each primer set by the formula E = 10-1/slope

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Supplementary Table 2.- Microbial and gene targets and bacterial strains and growth media utilized to develop standard curves for absolute quantification of different microbial groups by real-time quantitative PCR (qPCR).

Microbial group/species/gene Representative species and strains Mediuma Growth conditions

Bacteroidetes phylum Bacteroides thetaiotaomicron DSM 2079T GAM-Arg 37ºC Anaerobioc Clostridium leptum group Faecalibacterium prausnitzii DSM 17677T RCM 37ºC Anaerobioc Blautia coccoides DSM935 BHI-RCM (1:1) 37ºC Anaerobioc Clostridium coccoides group Blautia obeum DSM 25238 RCM 37ºC Anaerobioc Bifidobacterium spp. Bifidobacterium longum LMG1319.7 MRS Cys 37ºC Anaerobioc Enterobacteriaceae Escherichia coli DH10B LB 37ºC Aerobioc Lactobacillus spp. Lactobacillus plantarum WCFS1 MRS 37ºC Aerobioc Collinsella aerofaciens IPLA 37002 GAM-Arg 37ºC Anaerobioc Coriobacteriaceae Collinsella masseriensis IPLA 37003 GAM-Arg 37ºC Anaerobioc Atopobium cluster Eggerthella lenta IPLA 37001 GAM-Arg 37ºC Anaerobioc Eggerthella spp. E. lenta IPLA 37001 GAM-Arg 37ºC Anaerobioc Slackia isoflavoniconvertens DSM 22006T GAM-Arg 37ºC Anaerobioc Slackia spp. Slackia equalifaciens DSM 24851T GAM-Arg 37ºC Anaerobioc

tdr gene S. isoflavoniconvertens DSM 22006T GAM-Arg 37ºC Anaerobioc

Lactococcus garvieae L. garvieae IPLA 31405 GM17 32ºC Aerobioc Serratia marcescens S. marcescens SE41 LB 37ºC Aerobioc

aKey of the media: MRS, de Man-Rogosa-Sharpe; MRS Cys, MRS supplemented with 0.25% cysteine; BHI, Brain Heart Infusion; RCM, Reinforced Clostridium Medium; LB, Luria-Bertani; GAM-Arg, Gifu Anaerobic Medium supplemented with 0.5% arginine; GM17, M17 with 0.5% glucose.

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Article Transcriptional Regulation of the Equol Biosynthesis Gene Cluster in Adlercreutzia equolifaciens DSM19450T

Ana Belén Flórez 1,*, Lucía Vázquez 1, Javier Rodríguez 1, Begoña Redruello 2 and Baltasar Mayo 1

1 Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares s/n, Villaviciosa, 33300 Asturias, Spain; [email protected] (L.V.); [email protected] (J.R.); [email protected] (B.M.) 2 Servicios Científico-Técnicos, Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Paseo Río Linares s/n, Villaviciosa, 33300 Asturias, Spain; [email protected] * Correspondence: [email protected]; Tel.: +34-985-89-21-31

Received: 25 February 2019; Accepted: 30 April 2019; Published: 30 April 2019

Abstract: Given the emerging evidence of equol’s benefit to human health, understanding its synthesis and regulation in equol-producing bacteria is of paramount importance. Adlercreutzia equolifaciens DSM19450T is a human intestinal bacterium —for which the whole genome sequence is publicly available— that produces equol from the daidzein isoflavone. In the present work, daidzein (between 50 to 200 μM) was completely metabolized by cultures of A. equolifaciens DSM19450T after 10 h of incubation. However, only about one third of the added isoflavone was transformed into dihydrodaidzein and then into equol. Transcriptional analysis of the ORFs and intergenic regions of the bacterium’s equol gene cluster was therefore undertaken using RT-PCR and RT-qPCR techniques with the aim of identifying the genetic elements of equol biosynthesis and its regulation mechanisms. Compared to controls cultured without daidzein, the expression of all 13 contiguous genes in the equol cluster was enhanced in the presence of the isoflavone. Depending on the gene and the amount of daidzein in the medium, overexpression varied from 0.5- to about 4-log10 units. Four expression patterns of transcription were identified involving genes within the cluster. The genes dzr, ddr and tdr, which code for daidzein reductase, dihydrodaidzein reductase and tetrahydrodaidzein reductase respectively, and which have been shown involved in equol biosynthesis, were among the most strongly expressed genes in the cluster. These expression patterns correlated with the location of four putative ρ-independent terminator sequences in the cluster. All the intergenic regions were amplified by RT-PCR, indicating the operon to be transcribed as a single RNA molecule. These findings provide new knowledge on the metabolic transformation of daidzein into equol by A. equolifaciens DSM19450T, which might help in efforts to increase the endogenous formation of this compound and/or its biotechnological production.

Keywords: equol; daidzein; isoflavones; transcriptional regulation; equol-producing bacteria; Adlercreutzia equolifaciens

1. Introduction Epidemiological and interventional studies suggest that soy isoflavones are beneficial to human health inasmuch as they are associated with lessened menopause discomfort in women [1]. They have also been related with a fewer reduced risk of suffering hormone-dependent, cardiovascular and neurodegenerative diseases, and certain types of cancer within general

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Nutrients 2019, 11, 993 population [2–5]. In soy, isoflavones are found mostly as glycoside conjugates (daidzin, genistin, and glycitin) [6]. These β-glycosides have low estrogenic activity, and must be hydrolyzed into bioavailable isoflavone-aglycones (daidzein, genistein and glycitin, respectively) by cellular enzymes or enzymes from gut bacteria [7]. In the intestine, isoflavone-aglycones undergo further metabolic reactions generating compounds of greater biological activity or inactive metabolites [8]. Equol is a metabolite derived from the metabolism of daidzein, a major isoflavone predominantly found in soy-containing foods, produced exclusively by certain bacteria in the gut of humans and animals [9]. Equol is the isoflavone-derived compound with the strongest estrogenic [10] and antioxidant [11] activities. Based on its structural similarity to mammalian estrogens, equol may effectively bind to type β estrogen receptors but not those of type α, preventing menopausal symptoms without increasing the incidence of breast cancer [12]. All the animal species tested so far produce equol in response to soy or daidzein-containing diets [13]. However, it is produced by only 30–60% of humans [14,15]; it is likely that only these people will fully benefit from soy/daidzein consumption. This substantial inter-individual variation in equol production has been explained by differences in the composition of the gut microbiota [16]. Equol is an optically active molecule with two different enantiomers (R and S). However, the bacterial conversion of daidzein seems to produce only (S)-equol [13]. All but one of the equol- producing microbes isolated so far belong to the family Coriobacteriaceae [17]. Equol-producing strains have been identified for Adlercreutzia equolifaciens [18], Asaccharobacter celatus [19], Enterorhabdus mucosicola [20], Slackia equolifaciens [21], and Slackia isoflavoniconvertens [22]. Some other strains have been only identified at the genus level, and are named after their strain code, e.g., Eggerthella sp. YY7918 [23], Paraeggerthella sp. SNR40-432 [24], and Slackia sp. NATTS [25]. The single non-Coriobacteriaceae equol-producing strain identified so far is Lactococcus garvieae 20-92 [26]. Bacterial equol biosynthesis from daidzein proceeds via the intermediates dihydrodaidzein and tetrahydrodaidzein [27–29]. Gene cloning and genome analysis has revealed a gene cluster composed of eight open reading frames (ORFs) with a very similar genetic organization in all the strains studied so far, including L. garvieae [23,26,28,29]. Of these genes, three coding for a daidzein- dependent NADP reductase (converting daidzein into (R)-dihydrodaidzein) (dzr) a dihydrodaidzein reductase (converting (R)-dihydrodaidzein into trans-tetrahydrodaidzein) (ddr) and a tetrahydrodaidzein reductase (transforming trans-tetrahydrodaidzein to (S)-equol) (tdr) have been reported to be essential for equol production in S. isoflavoniconvertens [29]. These enzymes are induced by the presence of isoflavones, as demonstrated in both S. isoflavoniconvertens [29] and E. mucosicola [30]. However, neither the metabolic pathway involved in the metabolism of daidzein to equol nor its regulation are fully understood in any equol-producing species. Further knowledge of the control and regulation of the genes involved in equol production is required for its large-scale biotechnological production and for the design of strategies aimed to increase the endogenous production of equol. The present study reports the transcriptional analysis, by reverse-transcribed PCR and real- time quantitative PCR, of the genes and intergenic regions of the equol cluster in A. equolifaciens DSM19450T. In the presence of daidzein, all genes in the cluster were overexpressed. Although no evidence of distinct mRNA transcripts was seen, differences in the expression level of several groups of genes flanked by terminator-like sequences of different strength were observed. These results provide the first insight into the expression patterns in this bacterium of the genes involved in equol production in the presence and absence of daidzein.

2. Material and Methods

2.1. Bacterial Strain and Growth Conditions

Adlercreutzia equolifaciens DSM19450T, an equol-producing microorganism [18], was used as a prototype bacterium for studying the transcriptional regulation of equol biosynthesis. The strain was grown in Gifu anaerobic medium (GAM; Nissui Pharmaceutical, Tokyo, Japan) supplemented with 5 g/L arginine (Merck, Darmstad, Gemany) (GAM-Arg); 2% agar was added to the broth when

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Nutrients 2019, 11, 993 a solid formulation was required. Cultures were incubated at 37 °C under strict anoxic conditions (10% H2, 10% CO2, and 80% N2) in a Mac500 work station (Down Whitley Scientific, Shipley, UK). For equol production, an overnight culture of the strain in liquid GAM-Arg was used as a seed culture to inoculate at 10% fresh GAM-Arg broth supplemented with different concentrations of daidzein (0, 50, 100, 150, and 200 μM) (Toronto Research Chemicals, Toronto, Canada). Bacterial growth was monitored at 2 h time intervals for 24 h, measuring the optical density at 600 nm (OD600) in a spectrophotometer. The results are presented as the mean ± standard deviation of four independent cultures.

2.2. UHPLC Analysis Metabolites such as daidzein, dihydrodaidzein, and equol were identified and quantified by UHPLC using a reversed-phase Acquity UPLC™ BEH C18 1.7 μm column [31]. Samples (0.2 mL) were harvested from bacterial cultures every 2 h for 24 h, filtered through a 0.2 μm PTFE membrane (VWR, Radnor, PA, USA), and used directly in UHPLC analyses. Metabolite concentrations were estimated based on calibration curves prepared with known quantities of the corresponding standard compounds (all from Toronto Research Chemicals). Measurements were obtained for four independent cultures.

2.3. Nucleic Acid Extraction and cDNA Synthesis Genomic DNA was extracted and purified after growth in GAM-Arg broth for 24 h using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Following the manufacturer’s recommendations for Gram-positive bacteria, but using an in-house lysis buffer (20 mM Tris-HCl, 2mM EDTA, 1.2% Triton X-100, and 20 mg/mL lysozyme) supplemented with mutanolysin (0.1 U/μl) and RNAse (1.25 mg/mL). DNA was then eluted in sterile molecular biology grade water and stored at −20 °C until use. Total DNA was used as a template to determine the optimal amplification conditions and the efficiency of the primers used in succeeding reverse-transcription polymerase chain reaction (RT-PCR) and reverse-transcription quantitative PCR (RT-qPCR) assays. Total RNA was obtained after growing the bacterium in GAM-Arg medium supplemented with daidzein (concentrations ranging from 50 to 200 μM), using a culture without the isoflavone as a control. Samples (5 mL) were harvested from the cultures during the exponential growth phase, corresponding to an OD600nm of ~0.25 (approximately 8 h after inoculation). Cell pellets were obtained by centrifugation and stored at −20 °C until use. Total RNA was isolated from frozen pellets using the lysis method described above for DNA extraction, and purified using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Purified RNA was subjected to an additional treatment with DNase I (Qiagen) to eliminate any contaminating DNA. The absence of residual DNA in the samples was verified by real time PCR (qPCR) using the purified RNA as a template and the universal bacterial primers HDA1 and HDA2 of the 16S rRNA genes [32]. The concentration and purity of DNase-treated RNA samples were determined by measuring their absorbance at 260 nm (A260), and the ratio A260/A280 measured in an Epoch spectrophotometer (BioTek, Winooski, Vt., USA). The purified RNA was stored at −80 °C until required for complementary DNA (cDNA) synthesis. cDNA was produced from 0.25 μg of RNA using the iScript™ cDNA Synthesis Kit (Bio-Rad, Barcelona, Spain). Reverse transcriptase reactions were performed following the manufacturer’s instructions; i.e., one cycle of 25 °C for 5 min, 42 °C for 30 min and 85 °C for 5 min. The cDNA produced in this way was used as a template for qualitative and quantitative gene expression analyses. Unless otherwise indicated, all the reagents employed in nucleic acid extraction and purification were purchased from Sigma-Aldrich (St. Louis, CA, USA).

2.4. Gene Expression Analysis

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The qualitative expression of the genes within the equol cluster of A. equolifaciens DSM19450T (Supplementary Table S1) was analyzed by RT-PCR. Based on the genome sequence for the strain deposited in the public NCBI database (GenBank Accession no.: GCA_000478885.1) [33], oligonucleotide primers within the ORFs from AEQU_2235 through to AEQU_2223 were designed (Supplementary Table S2). For better comparison of the transcriptional signals, primers were designed to produce amplicons of around 500 bp. RT-PCR was performed using the Taq DNA Polymerase Master Mix Kit (Ampliqon, Odense, Denmark), adhering to the following amplification protocol: an initial step at 94 °C for 5 min, followed by 33 cycles of 94 °C for 30 s, 55–68 °C (depending on the primer pair; Supplementary Table S2) for 30 s, and 72 °C for 45 s, plus a final extension step at 72 °C for 7 min. Expression levels of the 16S rRNA genes were used as controls to confirm there were no major differences in cDNA concentration between samples. The RT-PCR products were separated by electrophoresis in 2% agarose gels, stained with ethidium bromide (0.5 μg/mL), and visualized under UV light using a G Box Chemi XRQ gel doc system (Syngene International, Bangalore, India). To elucidate the transcriptional organization of the above mentioned ORFs, new primers were designed within upstream and downstream ORFs to amplify the sequences of the complete intergenic regions (Supplementary Table S3). The PCR amplification conditions were the same as above. Additionally, the presence of inverted repeat sequences with a secondary structure that might provide transcriptional terminators was investigated using DNAMAN® v.5.2 software (Lynnon Biosoft, San Ramon, CA, USA). For selected genes, expression was quantified by RT-qPCR. Amplification was performed in an ABI PRISM 7500 thermocycler (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems). To ensure qPCR product size uniformity (≤90 bp) and melting temperature (60 °C ± 1 °C), primers (Supplementary Table S4) were designed using the algorithms provided by Primer Express v.2.0 software (Applied Biosystems). The efficiency of the primers was calculated based on the slope of a standard curve (Supplementary Table S4) and their specificity confirmed by detecting a single peak in the dissociation curve analysis of the amplicons. RT-qPCR was performed in triplicate for each target gene, with two independent experiments. To avoid variation among the samples in terms of the quantity and quality of cDNA, the expression of the housekeeping genes of A. equolifaciens glyceraldehyde-3-phosphate-dehydrogenase (GADPH) and the elongation factor Tu (EF-Tu) were used as controls. The results were recorded as the differential expression of a given gene with respect to the control sample (bacterial cultures grown without daidzein); i.e., by the 2−ΔΔCt method.

3. Results

3.1. Growth of A. equolifaciens DSM19450T with Daidzein The effect of daidzein on the growth of the bacterium was checked in parallel cultures grown in GAM-Arg supplemented with different concentrations of daidzein (0, 50, 100, 150, and 200 μM). Figure 1 shows the growth curves obtained under the given conditions over 24 h of incubation. No major differences were observed in the shape of the curves. Indeed, the profiles were almost identical until 10 h, after which, depending on the daidzein concentration, maximum growth occurred at 16 to 20 h of incubation, declining sharply thereafter. Compared to the control without daidzein, bacterial growth increased in the presence of all concentrations of daidzein tested (Figure 1). However, the maximum growth was reached in the culture supplemented with the lowest daidzein concentration (50 μM).

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Figure 1. Growth kinetic curves of A. equolifaciens DSM19450T grown in the presence of 50μM (green), 100μM (blue), 150μM (yellow) and 200μM (purple) of daidzein as compared to a control culture without daidzein (red).

3.2. Metabolism of Daidzein by A. equolifaciens DSM19450T The fate of the daidzein in the cultures, and its subsequent conversion into dihydrodaidzein and equol was followed over 24 h of incubation by UHPLC. Daidzein, dihydrodaidzein and equol were never detected in the absence of the isoflavone. Figure 2 shows the change in these three compounds over cultivation with the different amounts of daidzein. Depending on the initial amount, daidzein disappeared from the cultures between 6 and 10 h of incubation (Figure 2A). Dihydrodaidzein, which was monitored in parallel in the same cultures, showed maximum peaks at 4 to 6 h of incubation, reaching concentrations of 18, 24, 28 and 47 μM in the cultures with 50, 100, 150 and 200 μM of daidzein, respectively (Figure 2B). After this maximum, the dihydrodaidzein concentration decreased sharply after 10 h of cultivation, remaining thereafter constant at 5–10 μM. Equol reached their highest levels at about 10–12 h of incubation (Figure 2C), simultaneous with the stabilization of the dihydrodaidzein concentration; then, they remained constant until 24 h of incubation (Figure 2B). The maximum amount of equol produced was 14, 28, 42 and 54 μM for the cultures supplemented with 50, 100, 150 and 200 μM of daidzein respectively.

3.3. Identification of Daidzein-Induced Genes Comparison of the ORFs from the equol biosynthesis clusters of S. isoflavoniconvertens and A. equolifaciens showed a high degree of linear conservation of genes, although identity at the deduced amino acid level ranged between 39 and 82% (Supplementary Figure S1). To identify the gene products involved in daidzein metabolism, gene expression analysis of all the ORFs in the equol operon was performed by RT-PCR. For this, total RNA was isolated from exponential-phase cultures without daidzein (control) and with the different amounts of daidzein stated above. Transcription of the 16S rRNA gene was identical in all cultures, indicating that variations in gene expression due to differences in cDNA concentration or quality could be ruled out (Figure 3). In the absence of daidzein, no transcription of the genes was detected or was significantly lower than in cultures with daidzein (except for ORF AEQU_2224, which showed a similar pattern of expression with and without the isoflavone) (Figure 3). Further, the expression of ORFs between AEQU_2225 and AEQU_2233, which includes tdr, ddr and dzr, became higher with increasing daidzein in the culture medium, with the strongest expression recorded for 200 μM.

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Figure 2. Evolution of daidzein (A), dihydrodaidzein (B), and equol (C) in cultures of A. equolifaciens DSM19450T supplemented with 50μM (green), 100μM (blue), 150μM (yellow) and 200μM (purple) of daidzein.

Figure 3. Schematic organization and RT-PCR analysis of genes in the equol biosynthesis cluster of A. equolifaciens DSM19450T. Color key: in green, genes proved to be involved in the conversion of daidzein to equol, as reported for S. isoflavoniconvertens[29]; in orange, genes with putative activity in daidzein metabolism; in gray, other genes. The band intensity represents the level of transcription of each individual gene in the presence of the different daidzein concentrations tested (on the left). As a control, the expression of the 16S rRNA genes was used.

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3.4. Transcriptomic Analysis of the Equol Gene Cluster in the Presence of Daidzein RT-qPCR analysis was performed to quantify the effect of daidzein on the expression of ORFs in the equol operon. In the absence of daidzein, a very low basal expression of all the genes was recorded. Figure 4A shows the relative expression of each gene (with respect to the controls) in the presence of the tested daidzein concentrations (previously calibrated against EF-Tu expression). Highly similar results were obtained when expression was calibrated against the expression of the GADPH gene. In general, the quantification of the RT-qPCR transcripts corroborated the RT-PCR findings; i.e., the expression of all ORFs from AEQU_2235 to AEQU_2223 increased with increasing daidzein concentration (Figure 4A). However, these genes were not all overexpressed equally; indeed, four differential expression patterns were noted. The group of ORFs from AEQU_2225 to AEQU_2223 showed the lowest relative increases (up ~3.1–31.6 fold). A second expression pattern was recorded for ORF AEQU_2235, the relative overexpression of which (compared to controls) ranged between ~31.6 and 177.8-fold. A third expression pattern was recorded for ORFs between AEQU_2229 and AEQU_AEQ2226 (up ~17.7–1,000-fold), among which the dzr gene is included. Finally, the group of ORFs from AEQU_2234 to AEQU_2230, which includes tdr and ddr, showed the highest level of overexpression with increases from ~100 to 10,000-fold (2 to 4 log10 units) at the lowest and highest daidzein concentration, respectively.

Figure 4. Quantification of the effect of daidzein on the expression of genes involved in the equol biosynthesis gene cluster of A. equolifaciens DSM19450T by RT-qPCR (A). The graph shows the relative expression of the genes in the presence of 50 (green), 100 (blue), 150 (yellow) and 200 (purple) of daidzein in relation to gene expression of the reference condition (without daidzein) and after normalization with the expression of the elongation factor Tu housekeeping gene. Gene organization and transcriptional analysis by RT-PCR of the intergenic regions of the equol

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biosynthesis cluster of A. equolifaciens (B). Summary of the inverted repeat sequences resembling ρ- independent terminators and their respective free energy (Kcal/mol) (C). Terminators with the lowest (in bold) and highest free energy are indicated by filled and unfilled circles, respectively, in Panel B.

3.5. Transcriptional Organization of the Equol Biosynthesis Gene Cluster In silico analysis of the intergenic sequences in the equol biosynthesis gene cluster revealed eight putative ρ-independent terminator-like sequences in the intergenic regions. Four of the eight predicted terminators consisted of stems of 7–10 nucleotides separated by loops of two to fifteen nucleotides, upstream of the ORFs AEQU_2234, AEQU_2229, AEQU_2225, and AEQU_2224 (Figure 4B). All had a calculated Gibbs-free energy (ΔG) value of <–10 kcal/mol (Figure 4C), suggesting a strong transcription termination capacity. The position of these terminators agrees well with the gene expression patterns determined by RT-qPCR (the only exception being ORF AEQU_2225, for which no significant differences in the amount of transcripts were recorded with respect to the AEQU_2224 and AEQU_2223 ORFs). To confirm whether the predicted terminators related to different transcripts, the expression of the intergenic regions of all ORFs in the equol gene cluster was also analysed by RT-PCR (Figure 4B). Surprisingly, amplification was obtained for all intergenic regions, indicating that the operon was transcribed as a single RNA molecule of ~15 kbp long. Within the same expression patterns, genes located downstream of weak putative terminators identified (ΔG −7 to −8 kcal/mol) showed a little less transcription than those located upstream.

4. Discussion A transcriptional analysis of the ORFs and intergenic regions of the equol gene cluster of A. equolifaciens DSM19450T was undertaken in this work to identify the genetic elements involved in equol biosynthesis and its regulation. Although three reductase enzymes (daidzein-, dihydrodaidzein-, and tetrahydrodaidzein-reductase) have been described as essential in the conversion of daidzein to equol [34], few studies have examined the genetics and biochemistry of equol formation in equol-producing bacteria [26,29]. Proteomic analyses of S. isoflavoniconvertens grown with daidzein have shown the induction, in addition to the reductases, of five other proteins encoded by genes located upstream and downstream of those coding for these reductases [29]. Enzymes of this cluster are thought to be involved in equol production and might all be regulated in a coordinated manner [29]. Prior to starting the genetic analyses, the growth behavior of A. equolifaciens DSM19450T was examined in the presence of daidzein. Compared to control cultures, the presence of daidzein induced greater bacterial growth. However, the maximum growth was reached at the lowest daidzein concentration tested (50 μM), suggesting that, as seen in other intestinal bacterial species [35,36], the growth of A. equolifaciens might be modulated by the daidzein and/or equol concentration. The growth curves for A. equolifaciens with daidzein were very similar to those reported for S. isoflavoniconvertens [30] and E. mucosicola [37]. Nonetheless, A. equolifaciens in GAM- Arg grew much more than S. isoflavoniconvertens [30] or E. mucosicola [37] in BHI medium, or Slackia sp. NATTS in GAM with 1% glucose [21], suggesting that gut-dwelling bacteria might obtain additional energy from arginine via the arginine dihydrolase pathway [38]. The production of equol by A. equolifaciens increased with the amount of daidzein present, suggesting this compound induces its own metabolism. The induction of enzymes involved in daidzein conversion to equol has already been reported for E. mucosicola [30] and S. isoflavoniconvertens [29]. Generally speaking, A. equolifaciens DSM19450T metabolized most of the daidzein (>95%) present, but only under one third of this (27–29%) was converted into dihydrodaidzein. In contrast most dihydrodaidzein seems to be transformed into equol. All these metabolic steps occurred rather quickly, as the daidzein disappeared after just 10 h of incubation and the level of both dihydrodaidzein and equol remained constant thereafter. For other equol- producing bacteria; e.g., A. celatus do03 [19], Eggerthella spp. [39], S. isoflavoniconvertens [37], and Slackia NATTS [25], daidzein-to-equol conversion ratios ranging from 50 to 90% have been

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Nutrients 2019, 11, 993 reported. A. equolifaciens DSM19450T showed a daidzein-to-equol transformation ratio close to that reported for A. celatus (32%) [40]. The fact that all the daidzein disappeared but only a small amount was transformed into dihydrodaidzein strongly suggests that other daidzein-derived metabolites (not identified in this study) are also produced by A. equolifaciens DSM19450T. Indeed, the simultaneous production of equol and O-desmethylangolensin (O-DMA) has already been reported for Eubacterium ramulus Julong 601 [41]. The conversion of daidzein into novel, as yet un-detected derivatives, cannot be ruled out (the same has recently been reported for the conversion of genistein into 5-hydroxy-dehydroequol) [42]. The transcriptional analysis of the genes and their flanking intergenic sequences in the absence of daidzein revealed the low level constitutive expression of 13 genes in the A. equolifaciens cluster. The fact that the expression of most genes in the equol operon increased in the presence of daidzein further suggests that all are involved in its metabolism. Five genes within the cluster experienced similarly high induction; these genes coded for the dihydro- and tetrahydro-daidzein reductases, a dihydrodaidzein racemase, and the alpha and beta subunits of an electron flavoprotein (Supplementary Table S2). Beyond the reductases, other proteins of the cluster might be indispensable in, or have influence on, equol production. As such, a dihydrodaidzein racemase converting (R)-dihydrodaidzein into (S)-dihydrodaidzein has been demonstrated essential for efficient biosynthesis of equol in L. garvieae [43]. This protein is 48% homologous to that encoded by ORF AEQU_2234 in A. equolifaciens. The flavoprotein in the cluster (AEQU_2232 and AEQU_2233, alpha and beta subunits) might participate in the transfer of electrons required by the reductases (as demonstrated for other proteins of this family that funnel electrons into the electron transport chain) [44] or divert electrons from dehydrogenases to nitrogenases [45]). Recently, the structure of the daidzein reductase of Eggerthella was resolved to be a homo-octameric protein containing FMA, FAD and an aggregate of 4Fe-4S ions, all acting as cofactors [46]. The present results suggest that the ORFs surrounding the daidzein reductase gene (Supplementary Table S2; Supplementary Figure S1), all of which had a similar expression pattern, might encode proteins involved in the synthesis of some of the above cofactors. Still other genes might encode a two-component response regulator and glutamate synthase and dehydrogenase enzymes homologous to components of the isoflavone-induced NodVW system in Bradyrhizobium japonicum [47,48]. These proteins might detect and react to the presence of daidzein in the environment. Finally, genes encoding proteins reported to act as [Fe-S]-maturases (HydE enzymes in Asaccharobacter and Adlercreutzia [49]) might be responsible for the maturation of the flavine-dehydrogenase, as it contains an iron-sulphur binding domain (Supplementary Table S2; Supplementary Figure S1). Since no information on transcription termination in A. equolifaciens or other equol-producing species is currently available, an in silico search for terminator-like sequences was performed to help elucidate the organization and underlying regulation mechanisms within the equol biosynthesis gene cluster. Among the eight putative terminator sequences identified, four concurred with those proposed in S. isoflavoniconvertens [29]. The Gibbs free-energy values of the terminators correlated well with the different expression patterns determined by RT-qPCR. However, surprisingly, the transcriptional analysis of all intergenic regions revealed the whole operon to be transcribed as a single unit. This suggests that internal promoters/terminators within the cluster (producing shorter transcripts) might also exist, as recently demonstrated in operons from other bacteria [50]. These signals and/or other post-transcriptional regulatory circuits that modulate the efficiency of mRNA translation into protein might shape the production of equol by A. equolifaciens DSM19450T.

5. Conclusions

A. equolifaciens DSM19450T transforms daidzein in its growth medium into equol. However, only one third part of the daidzein added is converted into dihydrodaidzein and then into equol. The expression of the 13 contiguous genes tested was enhanced when the bacterium was incubated with daidzein, suggesting the expression of all these genes to be modulated by this isoflavone. Although the cluster was translated into a single RNA molecule, the genes were expressed under

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Nutrients 2019, 11, 993 four distinct expression patters, each under the control of different putative promoter and terminator signals. More research will be needed to discover all the elements of the daidzein metabolism pathways in A. equolifaciens and their involvement in equol production. This knowledge is thought to be pivotal in attaining large-scale biotechnological production of equol using this bacterium or its genetic machinery, and for designing strategies aimed at increasing its endogenous production.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1, Figure S1: Comparison of equol biosynthesis clusters from Slackia isoflavoniconvertens DSM22006T and A. equolifaciens DSM19450T. In S. isoflavoniconvertens, green-colored genes have been demonstrated to be essential for equol production; proteins from orange-colored genes have been found to be induced by daidzein; Table S1: Annotation of the open reading frames (orfs) within the equol biosynthesis gene cluster of A. equolifaciens DSM19450T; Table S2: Sequence, product size and annealing temperature of PCR primers used in gene expression analysis by RT-PCR; Table S3: Sequence, product size and annealing temperature of primers used to study gene expression of intergenic regions by RT-PCR; Table S4: Accuracy, efficiency and regression equation obtained for the amplification of equol cluster genes with the primers designed in this study for RT- qPCR analysis.

Author Contributions: A.B.F. and B. M. conceived the study. L.V. and J.R. performed most of the experiments and contributed to the discussion of the results. B.R. was involved in the chromatography analyses. B.M. provided material and human resources. A.B.F. and B.M. drafted and reviewed the manuscript. All authors reviewed and approved the final version.

Acknowledgments: This study was partially supported by projects from MINECO (AGL2014-57820-R) and Asturias Principality (IDI/2018/000114). LV was supported by a contract from the FPI Program of MINECO (BES-2015-072285). ABF was supported by a research contract from a CSIC project (201870E003).

Conflicts of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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ifcA ifcB ifcC tdr ddr ifcD dzr ifcE S. isoflavoniconvertens DSM22006T 11,255 bp

50% 71% 39% 58% 68% T 47% 51% 82% 43% 63% A. equolifaciens DSM19450 14,843 bp

AEQU_2235 AEQU_2233 AEQU_2231 AEQU_2230 AEQU_2228 AEQU_2227 AEQU_2225 AEQU_2224 AEQU_2223 AEQU_2234 AEQU_2232 AEQU_2229 AEQU_2226

Supplementary Figure 1.- Comparison of equol biosynthesis clusters from Slackia isoflavoniconvertens DSM22006T and A. equolifaciens DSM19450T. In S. isoflavoniconvertens, green-coloured genes have been demonstrated to be essential for equol production; proteins from orange-coloured genes have been found to be induced by daidzein (Schöder et al., 2013).

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Supplementary Table 1.- Annotation of the open reading frames (orfs) within the equol biosynthesis gene cluster of A. equolifaciens DSM19450T.

Locus_tag Strand Positiona Gene product

AEQU_2235 - 2791670-2792440 Two-component response regulator AEQU_2234 - 2791008-2791460 Putative dihydrodaizein racemase AEQU_2233 - 2790267-2790986 Electron transfer flavoprotein beta subunit AEQU_2232 - 2789323-2790237 Electron transfer flavoprotein alpha subunit AEQU_2231 - 2787796-2789259 Putative tetrahydrodaidzein reductase AEQU_2230 - 2786868-2787716 Putative dihydrodaidzein reductase AEQU_2229 - 2786295-2786774 Hypothetical protein AEQU_2228 - 2784304-2786232 Putative daidzein reductase AEQU_2227 - 2782920-2784233 Flavin-dependent dehydrogenase AEQU_2226 - 2782612-2782923 Putative ferredoxin AEQU_2225 - 2780557-2782395 Putative glutamate synthase AEQU_2224 - 2778770-2780563 Dehydrogenase AEQU_2223 - 2777598-2778668 Conserved hypothetical protein aIn Adlercreutzia equolifaciens DSM19450T genome (GenBank Accession no.: GCA_000478885.1).

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Supplementary Table 2.- Sequence, product size and annealing temperature of PCR primers used in gene expression analysis by RT-PCR.

Annealing Product length Gene Primers Secuencia 5´ - 3´ temperature (bp) (ºC)

35f GAGTTGGCATCATGATCTGCGATA EQU_2235 515 55 35r CGTCCATCAACAGGCAGAGAATCT 34f CGAGACCATGAGGGATTTCAACGA AEQU_2234 401 55 34r GCGTACAGGTCGATGCCCATGTC 33f GGTGACGAGGTGGTTGGTTTGT AEQU_2233 531 55 33r GGCAGGATCGGATCCATCGAACA 32f GCTGTCGCTTCGTCGGTCAGCAA AEQU_2232 456 68 32r CCGAAATACCCACCGACACGTAGA tdrf GATACCATCGATTTCCTCAAGGAT tdr 660 62 tdrr CGTTCTCAGCAAGACGGTCGATGT ddrf CTGGGCAAGCGATTGGAAGGTAA ddr 651 65 ddrr GATCGATAGCCTGCTGGGTGGTCT 29f CATTGGATTCGGAAGAGTTGTCA AEQU_2229 502 55 29r CAGGATCGAAGCCACTGATGGCTT dzrf GACCATCGAGGAGATTCACGAGTT dzr 702 55 dzrr CTCGCGATCGAACTGGTACAGTGT 27r GCAACATGACCATCGACTTCACCA AEQU_2227 537 62 27f GGATGGTGCTTGAAGTCTTCCAT 26r CACCTTGCCGAAGATCACGGTGAA AEQU_2226 255 60 26f CGACACGATGGTCTCCTCGCAGCA 25r GGTGAAGATGATCCGTAAGGACAA AEQU_2225 763 55 25f GAGCCTGCAGGCACATCATCTCGA 24r CGTTAACATTCCCACGCTGTGCT AEQU_2224 596 55 24f GCCACCTGCACCACGGTCTCCAC 23r CGAGGAAGGGTGGCAGGCAGGCTT AEQU_2223 615 68 24r CGACAAGTTCGGCATCACGACGTT

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Supplementary Table 3.- Sequence, product size and annealing temperature of primers used to study gene expression of intergenic regions by RT-PCR.

Annealing Intergenic Amplicon size Primers Sequence (5’ - 3’) temperature region (bp) (ºC)

35-34-F GAATCGTGTGCAGGCTGCTG 2235-2234a 332 60ºC 35-34-R CAGGTCGTTGAAATCCCTCA 34-33-F CAACATCGAAGTGGCTCTCA 2234-2233 283 60ºC 34-33-R GCACAAACCAACCACCTCGT 33-32-F GTTCGATGGATCCGATCCTG 2233-2232 375 60ºC 33-32-R GAACACGGGAGGCTGCAGCA 32-31-F CGACTTGAACAAGGTGGTT 2232-tdr 286 60ºC 32-31-R CTTCTGCTCGCTCGACTCGA 31-30-F GCAATATGCAGGTAGTCGACA tdr-ddr 348 60ºC 31-30-R GCTTACCTTCCAATCGCTTG 30-29-F GACCACCCAGCAGGCTATCGA ddr-2229 382 60ºC 30-29-R CGAACCAAGCCTCGATGACA 29-28-F GCAACATGCTGATCAACAAC 2229-dzr 340 60ºC 29-28-R CCATGGGCTGTCGCACGATG 28-27-F CGACGACATCGAGCAGATT dzr-2227 378 60ºC 28-27-R CCGTTGGCATACTTGTCGA 27-26-F CAGCAATCTGACGAAGCGTA 2227-2226 235 60ºC 27-26-R CTTCACCAGCTTCAGGAACT 26-25-F CATCTGCTGCGAGGAGACCA 2226-2225 359 60ºC 26-25-R GCCTCGACGAACGCTCCCAT 25-24-F CTGCCTGCGATGCGATGTGT 2225-2224 182 60ºC 25-24-R GATGCAGTTCAGATCCTTCA 24-23-F CCGACATTGCGAAGCTGTAC 2224-2223 396 60ºC 24-23-R GCAGATGTTCGTGAACTCGA aNumbers of the intergeneric regions refer to ORF numbers (AEQU_) or to their corresponding genes.

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Supplementary Table 4.- Accuracy, efficiency and regression equation obtained for the amplification of equol cluster genes with the primers designed in this study for RT-qPCR analysis.

Gen Primer Sequence (5´-3´) R² Regression equation Effiency Aequ_2235.qPCR-F CGCCTGAATCGTTCACTTCA AEQU_2235 0.9973 y = -3.3346x + 24.304 99.47% Aequ_2235.qPCR-R GTGTCGCTGTGCTGGTTCAA Aequ_2234.qPCR-F GTACTTCGAGCACAACCTGATGA AEQU_2234 0.9979 y = -3.3234x + 27.444 99.93% Aequ_2234.qPCR-R CGTTGAGAGCCACTTCGATGT Aequ_2233.qPCR-F CGCACGGTGGTGTTGGA AEQU_2233 0.9997 y = -3.3572x + 26.562 98.54% Aequ_2233.qPCR-R TTCAACACCCTTGATTTCCTCAA Aequ_2232.qPCR-F CGGTGCCAAGACCATCGT AEQU_2232 0.9988 y = -3.5962x + 29.539 100.55% Aequ_2232.qPCR-R GCAGGAACCACCTTGTTCAAG Aequ_2231.qPCR-F CAGCCACTATCGCGCAAACT tdr 0.9986 y = -3.2687x + 27.398 102.27% Aequ_2231.qPCR-R GATCCTTGAGGAAATCGATGGT Aequ_2230.qPCR-F CCACCCAGCAGGCTATCG ddr 0.9971 y = -3.255x + 27.107 102.87% Aequ_2230.qPCR-R CAGGTTGACCGCCGTGAT Aequ_2229.qPCR-F TCGGCACGATCATTTCGTT AEQU_2229 0.9988 y = -3.327x + 27.245 99.78% Aequ_2229.qPCR-R GACAAAGCCGCCGAACAC Aequ_2228.qPCR-F GGCCGTCTGGGCAAGTACTA dzr 0.9985 y = -3.3057x + 28.073 100.68% Aequ_2228.qPCR-R GGTCTCCGGCGTGGCATT Aequ_2227.qPCR-F GAGTCGGCTGGATCGAAGAAC AEQU_2227 0.9996 y = -3.2753x + 26.722 101.98% Aequ_2227.qPCR-R TTGGCATACTTGTCGAAAACCTT Aequ_2226.qPCR-F ATCACGGTGAACGTCGATGA AEQU_2226 0.9948 y = -3.2587x + 28,659 102.70% Aequ_2226.qPCR-R GCCGTGAAGCCGATATGC Aequ_2225.qPCR-F GTCGACCGCACCTATTTCGT AEQU_2225 0.9898 y = -3.3613x + 28.935 98.38% Aequ_2225.qPCR-R TGGCCATGATGACGGTCTT Aequ_2224.qPCR-F CTTCGACCGCGTCTTCGA AEQU_2224 0.9984 y = -3.4123x + 28.964 96.36% Aequ_2224.qPCR-R CCCAGGAACTCCACGAACTC Aequ_2223.qPCR-F CGCCAACGACTGCCACTAC AEQU_2223 0.9917 y = -3.3375x + 28.938 99.35% Aequ_2223.qPCR-R CAGAATCTGCTCACCGGTAAGG Aequ_Tuf.qPCR-F CACGCCGACTACGTGAAGAAC tuf 0.9933 y = -3.3272x + 28.897 99.78% Aequ_Tuf.qPCR-R CCGTCGGTAGCAGCGATAAC Aequ_gadpdh.qPCR-F AGCACGTGACCATGCTTTCC gadpdh 0.9982 y = -3.3168x + 27.253 100.21% Aequ_gadpdh.qPCR-R GGCCTCTACGACCACATCCA

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Article Cloning and expression of equol genes from Adlercreutzia equolifaciens in Escherichia coli and lactic acid bacteria

Short title: Cloning of equol genes

Abstract: In this work, four putative genes (racemase, tdr, ddr, and dzr) encoding key enzymes involved in equol production by Adlercreutzia equolifaciens DSM19450T were selected, codon-optimized, and synthesized preceded by a strong promoter from Lactococcus lactis, which drives expression in Escherichia coli and lactic acid bacteria (LAB). The synthetic DNA was cloned in E. coli in a general cloning vector pUC57 (pUC57-equol), and the whole construct cloned in pIL252, a low-copy- number vector with broad-host-range in Gram-positives (pIL252-pUC57-equol). The latter construct was purified from E. coli and then introduced by electroporation into strains of Lactobacillus casei and L. lactis. The production of equol from daidzein or dihydrodaidzein as a substrate by recombinant clones of these species was assessed in cultures. E. coli clones produced equol from both daidzein and dihydrodaidzein, although the amount at 24 h of incubation was higher for the pUC57-equol construct. A small amount of equol was produced by L. casei carrying the pIL252-pUC57-equol construct from dihydrodaidzein but not from daidzein. In contrast, L. lactis did not produce equol from any of the two substrates. E. coli clones harboring pUC57-equol could, therefore, be used for large-scale biotech production of equol. This will enable more intervention trials to be conducted to assess equol benefits in human health. More research is still required to produce equol from daidzein by LAB species using the genetic equol-associated machinery from A. equolifaciens.

Keywords: Soy isoflavones, daidzein, equol, Adlercreutzia equolifaciens, cloning, daidzein reductase, dihydrodaidzein reductase, tetrahydrodaidzein reductase

1. Introduction

Epidemiological and interventional studies from the family Eggerthellaceae of the suggest that consumption of soy and soy products Actinobacteria phylum (Salam et al., 2020). Within correlates with beneficial health effects on the this family, strains of species such as Adlercreutzia prevention and treatment of diseases and syndromes equolifaciens, Slackia isoflavoniconvertens, and such as postmenopausal symptoms, coronary-heart Slackia equolifaciens have been described as equol and neurological diseases, osteoporosis, and producers (Vázquez et al., 2017a; Braune et al., hormone-dependent cancers (Smeriglio et al., 2019; 2018). The equol biosynthesis in these bacteria takes Zaheer et al., 2017). The beneficial effects of soy are place through dihydrodaidzein and tetrahydrodaidzein attributed to its content in isoflavones and the intermediates via a process involving three reductases subsequent microbial-derived isoflavone metabolites. (Schröder et al., 2013; Tsuji et al., 2012). In Chemically, these compounds resemble the Lactococcus garvieae 20-92, the single well- endogenous 17-β-estradiol and are endowed with a characterized strain from the human gut producing hormonal-like activity (Franke et al., 2014; Vitale et equol and not belonging to the Eggerthellaceae al., 2013). Equol formed in the intestinal tract from family, a racemase has also been shown to be required daidzein by microbial components of the microbiota, for high equol production (Shimada et al., 2012). The is the isoflavone-derived compound with the highest racemase and the reductases are found in all equol- physiological activity (Mayo et al., 2019). However, producing strains characterized so far in a 10 kbp depending on origin and dietary habits, equol is only operon-like structure (Shimada et al., 2011; Schröder produced by 25-50% of the people; these might et al., 2013; Flórez et al., 2019). actually be the only that fully benefit from isoflavone Equol-producing bacteria are fastidious and consumption (Birru et al., 2016). extremely oxygen-susceptible species, which hinder Several equol-producing strains from the human the biotechnological production of equol to an intestine have been identified and characterized in the industrial scale with these organisms (Clavel et al., last decades (Mayo et al., 2019). Most of them belong 2014). Thus, the cloning and expression of the genetic to minority populations in the gut of anaerobic species machinery from equol producers in model organisms

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would be a rational approach to allow easy and demonstrated in E. coli. However, in the lactic acid inexpensive, large-scale, production of equol. This is bacterium L. casei equol production was only seen pivotal for carrying out a greater number of human when dihydrodaizein was used as a substrate, and trials to evaluate its efficacy and to extend the health equol was never produced by recombinant L. lactis benefits of equol to the general population, regardless clones. of whether they harbour in their gut equol-producing microbes. In this sense, the genes of L. garvieae 2. Material and Methods encoding the essential reductases (dzr, ddr, and tdr) 2.1. Plasmids, bacteria and culture conditions have been cloned and expressed in Escherichia coli (Shimada et al., 2010; Shimada et al., 2011). The Bacterial strains and plasmid vectors used in the cloning of equivalent genes from S. present work are summarized in Table 1. Escherichia isoflavoniconvertens and Eggerthella sp. YY7918 and coli DH10B was grown in Luria Bertani (LB) broth or the concomitant production of equol in the same host 2xTY medium with constant shaking at 37ºC. have been also reported (Schröder et al., 2013; Lactococcus lactis NZ9000 and Lactobacillus casei Kawada et al., 2016; Lee et al., 2017). BL23 were grown statically at 32ºC in M17 medium In this work, we are reporting on the cloning and (Biokar, Beauvais, France) supplemented with 1% expression in E. coli, Lactobacillus casei, and (wt/vol) glucose (GM17), and MRS (Merck, Lactococcus lactis of a synthetic DNA fragment Darmstadt, Germany), respectively. Agar (2% wt/vol) based on sequences of the equol operon from A. was added to the media when solid media were equolifaciens DSM19450T. The construct contains required. Liquid and solid media were supplemented three genes encoding the essential reductases required with appropriate antibiotics for selection of the -1 for equol biosynthesis (dzr, ddr, and tdr) under their transformants and plasmid maintenance, 100 μg mL -1 own promoter preceded by the racemase gene under ampicillin and 300 μg mL erythromycin for E. coli, -1 the control of a strong constitutive promoter from L. and 2.5 or 5 μg mL of erythromycin for L. lactis and lactis. Transformation of daidzein into equol was L. casei.

Table 1.- Bacterial strains, synthetic DNA, and plasmids used in the present study.

Strain, synthetic DNA, Relevant genotype, description or properties Reference or source plasmid

Strains F–, mcrA, Δ(mrr-hsdRMS-mcrBC), φ80lacZΔM15, ΔlacX74, recA1, ThermoFisher Escherichia coli DH10B endA1, araD139, Δ(ara-leu)7697, galU, galK, λ–, rpsL(StrR), nupG Scientific L. lactis subsp. cremoris MG1363 derivative pepN::nisRK; plasmid Lactococcus lactis NZ9000 Kuipers et al. (1998) free Acedo-Félix and Lactobacillus casei BL23 Plasmid free strain Pérez-Martínez(2003) E. coli pUC57 E. coli DH10B carrying pUC57; Ampr This study

L. lactis pIL252 L. lactis NZ9000 carrying pIL252; Emr This study E. coli DH10B-pUC57- E. coli DH10B carrying pUC57-equol; Ampr This study equol E. coli DH10B-pIL252- E. coli DH10B carrying pIL252-pUC57-equol ; Ampr This study pUC57-equol L. lactis NZ9000-pIL252- L. lactis NZ9000 carrying pIL252-pUC57-equol; Emr This study pUC57-equol L. casei BL23-pIL252- L. casei BL23 (pLZ15-) carrying pIL252-pUC57-equol; Emr This study pUC57-equol

(Continued)

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Table 1.- Continued. Strain, synthetic DNA, Relevant genotype, description or properties Reference or source plasmid

Plasmids ThermoFisher pUC57 pUC19-derived general cloning vector; Ampr Scientific Recombinant plasmid containing the synthetic DNA carrying the pUC57-equol equol genes racemase, tdr, ddr, and dzr preceded by P59 promoter This study cloned in pUC57; Ampr Low copy-number cloning vector for Gram-positives, based on the Simon and Chopin pIL252 replicon of pAMβ1 from Enterococcus faecalis; Emr (1988) Recombinant plasmid containing pUC57-equol cloned in pIL252; pIL252-pUC57-equol This study Ampr, Emr

Synthetic DNA A DNA segment of 5,206 nucleotides long (Suppplementary Figure 1), including four ORFs based on the genome sequence of Adlercreutzia equolifaciens DSM19450T: racemase (AEQU_2234), - equol This study tetrahydrodaidzein reductase (tdr, AEQU_2231), dihydrodaidzein reductase (ddr, AEQU_2230) and daidzein reductase (dzr, AEQU_2228).

Ampr, resistance to ampicillin; Emr, resistance to erythromycin.

2.2. Design of synthetic DNA The DNA sequence of the equol biosynthesis Russell (2001). DNA from agarose gels was purified gene cluster from the genome of Adlercreutzia using the GFX PCR DNA Gel Band Purification kit equolifaciens DSM19450T was retrieved from (GE Healthcare Biosciences, Buckinghamshire, GenBank (NC_022567.1; Maruo et al., 2008). The UK). The In-Fusion cloning kit (Clontech, Mountain sequence of four ORFs thought to be involved in the View, CA, USA) was used according to the synthesis of equol, including the racemase gene manufacturer's instructions. Restriction (AEQU_2234), and those encoding the putative endonucleases (Takara, Otsu, Shiga, Japan) and T4 downstream reductases, tetrahydrodaidzein DNA ligase (Invitrogen, Carlsbad, CA, USA), were reductase (tdr, AEQU_2231), dihydrodaidzein used as recommended by their manufacturers. reductase (ddr, AEQU_2230), and daidzein Electrocompetent cells of E. coli DH10B were reductase (dzr, AEQU_2228) was codon-optimized prepared as reported by Sambrook and Russell for expression in E. coli, assembled leaving the (2001). Electrocompetent L. casei and L. lactis cells native intergenic signals, and located under the were prepared according the procedure by Holo and control of the constitutive promoter P59 from L. Nes (1989). Electrotransformation (electroporation) lactis (van der Vossen et al., 1987). To facilitate was done by using a Gene Pulser apparatus (Bio- cloning, some restriction enzymes sites were Rad, Richmond, CA, USA) following standard removed or added, and flanking 20-bp sequences protocols for Gram-negative and Gram-positive identical to those adjacent to the multiple cloning bacteria. White/blue screening for pUC57 in E. coli site of pUC57 were annexed (Supplementary Figure was performed on LB plates supplemented with 1). A final synthetic DNA consisting of 5,206 bp appropriate antibiotics, and 5-bromo-4-chloro-3- long sequence was synthesized and purified at indolyl-ß-D-galactopyronoside (20 mg/mL; X-Gal, Synbio Technologies (Monmouth Junction, NJ, Sigma-Aldrich, St. Louis, CA, USA) and isopropyl- USA). ß-D-thiogalactopyranoside (0.5 M, Sigma-Aldrich). Transformants of L. lactis and L. casei were selected 2.3. DNA manipulation and cloning onto GM17 or MRS agar plates with erythromycin. General procedures for DNA manipulation were Plasmid DNA from E. coli was isolated and purified followed essentially as described by Sambrook and as described by Sambrook and Russell (2001).

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Bam HI (611) lacZ NheI (139) MC Nhe I (655) lac repressor Bam HI (5045) r Eco RI (10) NheI (2200) Bam HI (3058) HindIII (5151) Am pUC57 Sal I (4) BamHI (2139) Nhe I (3110) Sal I (5158) 2,710 bp

tdr ddr dzr pMB1 P59 ori racemase equol 5,206 bp Digestion with In-fusion EcoRI-HindIII ligation

EcoRI (405) r Am P59 racemase

repD pMB1 r Em ori

pUC57-equol tdr pIL252 7,795 bp 4,698 bp

repE Eco RI (2876) Eco RI (2818) ddr dzr Digestion with Digestion with EcoRI T4 ligation EcoRI

r Eco RI (405) Am

pMB1 r ori Em

repD pIL252-pUC57 -equol dzr 12,435 bp

repE

ddr Eco RI (5045)

P59 tdr racemase

Figure 1.- Physical map of the plasmid constructs obtained in this work. The synthetic DNA cloned in pUC57 carries four equol-related genes from Adlercreutzia equolifaciens DSM19450T: the racemase gene, and the genes tdr, ddr, and dzr, which encode a racemase, and the tetrahydrodaidzein, dihydrodaidzein, and daidzein reductases, respectively, preceded by promoter P59 from Lactococcus lactis subsp. cremoris Wg2. The colour key of the genes: in red, antibiotic resistance genes (Amr, ampicillin resistance; Emr, erythromycin resistance); in light blue, origin of replication or genes encoding replication proteins; in green, genes involved in equol production; in purple, promoter P59. Relevant restriction enzyme sites are also indicated. Molecules are not drawn to scale.

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Plasmids from L. casei and L. lactis was isolated by above conditions. This isoflavone concentration did a modification of the procedure of O'Sullivan and not affect growth of the cells from controls or Klaenhammer (1993), involving the addition to the clones. Culture supernatants were sampled after lysis buffer before phenol/chloroform extraction of 4 inoculation (T0), at 8 h of incubation (T8), and at 24 μL of mutanolysin (5 U mL-1) and 20 μL of h (T24), and analyzed by HPLC for detection and proteinase K (20 mg mL-1). Constructs were verified quantification of daidzein and its metabolites by digestion with restriction enzymes, sequencing dihydrodaidzein and equol. The results are and sequence analysis. summarized in Table 2. In the control cultures, only the supplemented isoflavone, either daidzein or 2.4. Identification and quantification of isoflavones dihydrodaidzein, were recovered. The recovery of A single colony of each clone was picked and daidzein was very low (in between 15 to 20% of the grown in 5 ml of appropriate liquid medium and supplemented amount), while that of incubated overnight under the species-specific dihydrodaidzein was almost complete (about 97%). conditions stated above. These cultures were then At 24 h of incubation, cultures of E. coli with the used for inoculating at 10% fresh medium pUC57-equol construct transformed a major part of supplemented independently with 200 µM of the daidzein into equol (about 90%). Surprisingly, daidzein or dihydrodaidzein (both from LC equol production by this construct was only partial laboratories, Woburn, MA, USA). As negative when dihydrodaidzein was used as a substrate controls, plasmid-free bacterial hosts and strains (≈15%), and almost half of the amount was carrying empty pUC57 and pIL252 plasmids were recovered as dihydrodaidzein. However, large cultured in the same conditions. Bacterial growth in variability in the transformation of daidzein and the presence of daidzein or dihydrodaidzein was dihydrodaidzein to equol was occasionally measured monitored spectophotometrically by measuring the in certain clones, including some that did not absorbance at 600 nm (A600) using uninoculated produce any equol from these substrates. Odd medium as a blank. Isoflavones and their values, which were not taken into account to metabolites were detected and quantified after calculate the averages, correlated with filtering through a 0.2 µm PTFE membrane (VWR, reorganizations and/or deletions in the constructs as Radnor, PA, USA) by high performance liquid revealed by restriction DNA analysis. Whatever the chromatography (HPLC) according to Redruello et case, these results demonstrated that the synthetic al. (2015). Metabolite quantification was determined genes based on those of A. equolifaciens were against calibration curves prepared using functional in E. coli and lead to the synthesis of commercial standards (all from LC laboratories). equol from both daidzein and dihydrodaidzein. Purified plasmid DNA of a pUC57-equol 3. Results construct, with their equol genes oriented in a The synthetic DNA fragment of 5,206 bp counterclockwise manner to the betalactamase gene (Supplementary Figure 1) was cloned in pUC57 by (Figure 1), and pIL252 were independently digested the In-Fusion technique giving rise to a recombinant with EcoRI. The molecules were extracted and construct, pUC57-equol (Figure 1). After purified from an agarose gel, ligated with T4 DNA homologous recombination between the 20 bp end ligase and the reaction mixture was electroporated sequences of the synthetic DNA and the linearized into competent E. coli DH10B cells. Transformants pUC57, the whole multi-cloning site of the vector carrying the pIL252-pUC57-equol construct (Figure was completely replaced by the synthetic construct, 1) were obtained and verified as above. Colonies of with a new sequence of 5,166 bp. The cloning this construct were cultured as before in the presence mixture was introduced by electrotransformation of daidzein and dihydrodaidzein (Table 2). At T24, into competent cells of E. coli DH10B, and equol was recovered from the supernatants when transformants were verified by restriction enzyme either daidzein or dihydrodaidzein were added as a analysis and sequencing. Single colonies of several substrate, indicating again that the biochemical pUC57-equol clones were picked at random and pathway for equol production was functional in the inoculated in 5 mL of LB medium with ampicillin new construct. However, the production was much (for plasmid maintenance), and 200 µM of daidzein lower than that from the pUC57-equol clones. From or dihydrodaidzein, and incubated under the specific both daidzein and dihydrodaidzein substrates the

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latter compound was the main metabolite and only Except for L. garvieae 20-92, all intestinal equol- about 5% of the substrates was recovered as equol. producing bacteria belong to the Eggerthellaceae DNA from the pIL252-pUC57-equol construct family whose members are strict anaerobes and have was then used to electroporate competent cells of L. abundant nutritional requirements (Salam et al., casei BL23 and L. lactis NZ9000. Transformants 2020), qualities that hinder their application for were recovered from the two strains and the equol production at an industrial scale, as the constructs analyzed again by restriction enzyme biotechnological synthesis of equol by these digestion and sequencing. Colonies of clones in both nutritive fastidious and strictly anaerobic bacteria L. casei and L. lactis were grown in 5 mL of MRS would require long culture times and expensive and GM17 medium, respectively, with erythromycin equipment (Clavel et al., 2014). To overcome the and incubated overnight under their optimal difficulties of anaerobic culturing in industrial respective growth conditions. These cultures were fermentations, “aerobic domestication” of the then used for inoculating at 10% fresh liquid media producing strains has been suggested (Zhao et al., supplemented independently with 200 µM of 2011). Another rational approach to circumvent daidzein or dihydrodaidzein. As a negative control, these challenges would be the cloning and plasmid-free cells of the recipient strains were expression of the equol production machinery in cultured under the same conditions. Optical density easily cultivable heterologous hosts (Tsuji et al., monitoring showed daidzein and dihydrodaidzein in 2012; Schröder et al., 2013). Large-scale biotech the cultures had no influence in bacterial growth of production of equol will brake equol shortage L. casei or L. lactis cells of recombinants and enabling more intervention trials to be conducted for controls. Supernatants of the cultures were sampled assessing the health benefits of equol (Selvaraj et al., for detection and quantification of isoflavone 2004). These interventions could help to clarify the metabolites at T0, T8, and T24 (Table 2). Again the inconsistent results of many studies and metaanalysis recovery of daidzein from supernatants was rather on the evidence of isoflavone-rich diets to reduce the variable and low (between 10 to 35%), as compared risk of a number of syndromes and chronic diseases to the recovery of dihydrodaidzein (>90% in most (Harland and Haffner, 2008; Bolaños et al., 2010; cultures). In the lactic acid bacteria (LAB) Wei et al., 2012; He and Chen, 2013; Liu et al., 2014; transformants carrying the pIL252-pUC57-equol Zhou et al., 2015; Fang et al., 2016; Akhlaghi et al., construct, equol was only detected at T24 in the 2017). Among other factors, those discrepancies have cultures of L. casei BL23 with dihydrodaidzein as a traditionally been attributed to a large part of the substrate. Transformation of DNA from the human population with a non-producing equol recombinant L. casei and L. lactis clones back to E. phenotype (Daily et al., 2019), as this isoflavone- coli proved the constructs to be fully functional in derived metabolite has the strongest hormonal activity this host, producing an amount of equol equivalent and the highest antioxidant action (Setchell and Cole, to that measured initially (10-12 µM). 2006). The production of equol in cultures by the 4. Discussion recombinant clones was tested using as substrates daidzein, the original isoflavone precursor of equol, In this work, a DNA fragment containing four and dihydrodaidzein, an intermediate compound in codon-optimized genes encoding enzymes involved the equol biosynthesis (Mayo et al., 2019). Daidzein in equol production based on sequences from A. and its metabolites were then identified and equolifaciens DSM19450T, preceded by a promoter quantified by HPLC analysis. As it has been from L. lactis, was synthesized and cloned in E. coli. reported before (Vázquez et al., 2017b), daidzein or To our knowledge, this is the first report on the dihydrodaidzein did not significantly affect growth cloning and expression of equol genes from A. of the different hosts and clones. Daidzein was equolifaciens in a heterologous host. One of the poorly recovered from the culture supernatants, constructs was cloned in a plasmid cloning vector which agrees well with the low recovery of this having a replicon acting on Gram-positives; this was isoflavone from biological samples (urine), as has. then transferred to model strains the LAB species L. lactis and L. casei.

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Table 2.- Daidzein and daidzein-derived metabolites in daidzein- or dihydrodaidzein-supplemented cultures of recombinant Escherichia coli DH10B, Lactobacillus casei BL23 and Lactococcus lactis NZ9000 cells harbouring equol-associated genes from Adlercreutzia equolifaciens DSM19450T.

Daidzein and daidzein-derived metabolite (µM; mean±SD) Sampling time (h) Strain/construct T0 T8 T24 Dzen Dh-dzen Equol Dzen Dh-dzen Equol Dzen Dh-dzen Equol

Daidzein (200 µM) E. coli 25.8* - - 9.3* - - 44.5±6.9 - - E. coli pUC57-equol 34.1±1.1 - - 8.2±0.9 105.8±2.3 0.1±0.2 - - 179.6±38.6 E. coli pIL252-pUC57-equol 24.9* - - 10.4±1.0 114.2±14.2 2.5±0.4 9.4±2.6 132.1±11.3 11.1±2.1

L. casei 67.9* - - 23.5* - - 35.4* - - L. casei pIL252-pUC57-equol 70.9±1.7 - - 30.9±5.1 - - 38.7±9.6 - -

L. lactis 30.7* - - 31.9* - - 26.8* - - L. lactis pIL252-pUC57-equol 23.6±2.4 - - 23.4±2.6 - - 29.0±0.6 - -

Control (pUC57) nd nd nd nd nd nd 147.4* - - Control (pIL252) nd nd nd nd nd nd 255.7* - -

Dihydrodaidzein (200 µM) E. coli - 193.6* - 2.7* 187.2* - - 119.9* - E. coli pUC57-equol - 181.0±2.4 - 12.9* 146.2±32.3 0.6* - 84.1±5.9 34.9±2.1 E. coli pIL252-pUC57-equol - 98.8±25.8 - 4.7±0.2 155.4±4.9 2.3±0.3 - 149.4±8.2 12.0±2.7

L. casei - 191.2* - - 190.4* - - 176.6* - L. casei pIL252-pUC57-equol - 136.5±3.1 - - 126.1±6.4 - - 48.6±2.9 4.8±2.2

L. lactis - 188.6* - - 191.3* - - 204.2* - L. lactis pIL252-pUC57-equol - 233.7±37.7 - - 201.7±12.6 - - 182.5±1.0 -

Control (pUC57) nd nd nd nd nd nd - 175.2* - Control (pIL252) nd nd nd nd nd nd - 148.6* - nd, not determined; -, not detected or below the LoQ; *data from a single representative experiment.

140 CAPÍTULO 3

been reported elsewhere (Redruello et al., 2015). fact that a small amount of equol was formed by L. This might result from an interaction of the casei from dihydrodaidzein indicates that the compound with components of the media or from a dihydrodaidzein reductase and the rapid absorption of the isoflavone by the bacterial tetrahydrodaidzein reductase genes were correctly cells (Wang et al., 2004; Obara et al., 2019). In transcribed, translated and the enzymes were active. contrast, a majority of the added dihydrodaidzein In contrast, equol from either substrate was never was recovered with the same method, suggesting detected in L. lactis cultures. The P59 promoter of L. this compound is more stable or has less interaction lactis has proven to drive expression of homologous with media components. and heterologous proteins in both E. coli (van der The two constructs, pUC57-equol and pIL252- Vossen et al., 1987), L. lactis (Que et al., 2000; pUC57-equol, were shown to drive equol production Quistián-Martínez et al., 2010), and L. casei (Gold in E. coli from either daidzein or dihydrodaidzein as et al., 1996) strains. However, some intergenic a substrate. This was not surprising, as expression in regions containing native A. equolifaciens signals E. coli of equol genes from different equol- might have not been appropriately identified by the producing species has been repeatedly reported transcription and translation machinery of LAB. (Shimada et al., 2010; Shimada et al., 2011; Transformation of the recombinant DNA from L. Schröder et al., 2013; Kawada et al., 2016). As in casei and L. lactis back into E. coli restored the this work, a low equol fermentation yield in E. coli capability of equol production, which indicates that has been reported in other works (Lee et al., 2016; the constructs were functional. Considering that the Li et al., 2018). This has been attributed to the low equol operon of L. garvieae seems to have been solubility of isoflavones (and thus daidzein) in acquired by horizontal transference from an aqueous systems (del Rio et al., 2013), a problem Eggerthellaceae species (Shimada et al., 2010; that has been recently overcome by adding Shimada et al., 2011), expression of equol genes in hydrophilic polymers to the cultures (Lee et al., other LAB strains should be feasible. Optimization 2018). The fact that the recombinant E. coli cultures of codon usage for E. coli might be a further source carrying the pUC57-equol construct produced more of poor or no expression of some genes in LAB. The equol than those carrying pIL252-pUC57-equol can GC content of L. lactis (34%) is rather lower than be due to the larger size of the latter construct that of E. coli and L. casei, which is quite similar leading to a reduced copy number. However, a lower (≈50%). However, the genetics of LAB (Bintsis, structural stability of the larger construct cannot be 2018) can still be rather different to that of E. coli, excluded. In fact, instability of the constructs was thus requiring specific translation and transcription occasionally seen with pUC57-equol, which might signals. account for the large statistical deviations in the The production of equol by recombinant BAL isoflavone metabolites. Indeed, instability of some species and strains could serve as an alternative equol genes in E. coli have already been reported, and source of “safe” equol due to the qualified mutations in the ddr gene (encoding the presumption of safety (QPS) status of these bacteria dihydrodaidzein reductase) has been reported to (EFSA BIOHAZ Panel, 2020). Production of equol contribute to clone stabilization, thus attaining higher by genetically modified LAB capable of developing equol production in cultures (Lee et al., 2016). properly in soybean extracts (Delgado et al., 2019), Mutations in housekeeping genes of the E. coli host would make possible the production of equol in have also been revealed to contribute to the stability fermented soybean products leading to equol- of the constructs, which brings to increase equol enriched foods. In spite of the current legal production (Lee et al., 2016). A mutation in ydiS constraints of the use of recombinant gene, considered as an equol resistant gene, has been microorganisms in food (EFSA GMO Panel, 2011), shown to overcome the equol inhibitory activity equol-enriched functional foods could meet the against E. coli (Li et al., 2018). needs of those individuals of the population that Neither the recombinant strains of L. casei nor cannot produce equol endogenously. those of L. lactis produced dihydrodaidzein or equol In conclusion, synthetic genes for equol from daidzein, which was recovered untransformed production based on sequences from A. from the LAB cultures at all sampling points. The equolifaciens were successfully cloned in E. coli and

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LAB species. Concomitant equol production from Actinobacteria, eds. Rosenberg, E., DeLong, E.F., daidzein during growth by the recombinant clones in Lory, S., Stackebrandt, E., and Thompson, F. (Eds.). cultures was achieved in E. coli. However, pp. 201-238. Springer-Verlag, Berlin. production of equol from daidzein in L. casei and L. Daily, J.W., Ko, B.S., Ryuk, J., Liu, M., Zhang, W., and lactis strains were never detected. Nonetheless, in Park, S. 2019. Equol decreases hot flashes in the former organism small amounts of equol were postmenopausal women: A systematic review and meta-analysis of randomized clinical trials. J. Med. formed from dihydrodaidzein. Further attempts will Food 22, 127-139. be made to express the equol genes of A. Del Rio, D., Rodriguez-Mateos, A., Spencer, J.P., equolifaciens by using newly designed synthetic Tognolini, M., Borges, G., and Crozier, A. 2013. genes under the control of LAB-specific Dietary (poly)phenolics in human health: structures, transcription and translations signals. bioavailability, and evidence of protective effects against chronic diseases. Antiox. Redox Signaling 18, Acknowledgments 1818-1892. This study was funded by projects from the Delgado, S., Guadamuro, L., Flórez, A.B., Vázquez, L., Spanish Ministry of Economy and Competitiveness and Mayo, B. 2019. Fermentation of commercial soy beverages with lactobacilli and bifidobacteria strains (MINECO) (AGL2014-57820-R) and the featuring high β-galactosidase activity. Innov. Food Principality of Asturias (IDI/2018/000114). LV was Sci. Emerg. Technol. 51, 148-155. supported by a research contract from the FPI EFSA BIOHAZ Panel. 2020. Update of the list of QPS‐ Program and from MINECO (BES-2015-072285). recommended biological agents intentionally added to food or feed as notified to EFSA 11: suitability of References taxonomic units notified to EFSA until September Acedo-Félix, E., and Pérez-Martínez, G. 2003. Significant 2019. EFSA J. 18, 5965. differences between Lactobacillus casei subsp. casei EFSA GMO Panel. 2011. Guidance on the risk assessment ATCC 393T and a commonly used plasmid-cured of genetically modified microorganisms and their derivative revealed by a polyphasic study. Int. J. Syst. products intended for food and feed use. EFSA J. 9, Evol. Microbiol. 53, 67-75. 2193. Akhlaghi, M., Zare, M., and Nouripour, F. 2017. Effect of Fang, K., Dong, H., Wang, D., Gong, J., Huang, W., and soy and soy isoflavones on obesity-related Lu, F. 2016. Soy isoflavones and glucose metabolism anthropometric measures: A systematic review and in menopausal women: A systematic review and meta- meta-analysis of randomized controlled clinical trials. analysis of randomized controlled trials. Mol. Nutr. Adv. Nutr. 8, 705-717. Food Res. 60, 1602-1614. Bintsis, T. 2018. Lactic acid bacteria as starter cultures: Flórez, A.B., Vázquez, L., Rodríguez, J., Redruello, B., An update in their metabolism and genetics. AIMS and Mayo, B. 2019. Transcriptional regulation of the Microbiol. 4, 665-684. equol biosynthesis gene cluster in Adlercreutzia T Birru, R.L., Ahuja, V., Vishnu, A., Evans, R.W., equolifaciens DSM19450 . Nutrients 11 pii: E993. Miyamoto, Y., Miura, K., Usui, T., and Sekikawa, A. Franke, A.A., Lai, J.F., and Halm, B.M. 2014. Absorption, 2016. The impact of equol-producing status in distribution, metabolism, and excretion of modifying the effect of soya isoflavones on risk isoflavonoids after soy intake. Arch. Biochem. factors for CHD: a systematic review of randomised Biophys. 59, 24-28. controlled trials. J. Nutr. Sci. 5, e30. Gold, R.S., Meagher, M.M., Tong, S., Hutkins, R.W., and Bolaños, R., Del Castillo, A., and Francia, J. 2010. Soy Conway, T. 1996. Cloning and expression of the isoflavones versus placebo in the treatment of Zymomonas mobilis “production of ethanol” genes in climacteric vasomotor symptoms: Systematic review Lactobacillus casei. Curr. Microbiol. 33, 256-260. and meta-analysis. Menopause 17, 660-666. Harland, J.I., and Haffner, T.A. 2008. Systematic review, Braune, A., and Blaut, M. 2018. Evaluation of inter- meta-analysis and regression of randomised controlled individual differences in gut bacterial isoflavone trials reporting an association between an intake of bioactivation in humans by PCR-based targeting of circa 25 g soya protein per day and blood cholesterol. genes involved in equol formation. J. Appl. Microbiol. Atherosclerosis 200, 13–27. 124, 220-231. He, F.J., and Chen, J.Q. 2013. Consumption of soybean, Clavel, T., Lepage, P., and Charrier, C. 2014. The family soy foods, soy isoflavones and breast cancer Coriobacteriaceae. In The Prokaryotes- incidence: Differences between Chinese women and

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MATERIAL SUPLEMENTARIO gttgtaaaacgacggccagt (sequence from pUC57)

SalI EcoRI CAGTCGACGAATTCAATATTCAGGTTTAAGAGACAATAAGAAAAAAAGATTGAAAAAAG -35 -10 TGACATTAAATTCTTGACAGGGAGAGATAGGTTTGATAGAATATAATAGTTGTCGCGAG AGACG (P59 from Lactococcus lactis subsp. cremoris Wg2) NheI AAGAAAGGAGGTATGCTAGC (sequence from Adlercreutzia equolifaciens DSM 19450T)

ATGAAGGGCAAGCTGATTCGCGTGTGCGTCGTGGCCAATGACGCGGCCGAGACCATGAG GGATTTCAACGACCTGTTCGGGGTCGAGTTCTACGGGCCGTTTGATGACGAGCATGTGA AGCTGAAGGTGGCGCTGCCGCGCAGCGGCGGCATGGAGGTGTCCTCCCCGACGGCCGCC GACGACCCCATCGGCTTCTCGCAGTATCTGGCCGCTCACGGCGAGGGCATCAAGGGCAT CGCCTTGCGCGTGGACAACATCCAGGAGGCCATGGACGAGCTGAAGGCCAAGGGCGTCG AGCTTGGCGTGTACTTCGAGCACAACCTGATGAAGGAGTGCATCATCCCCCCGCAGCCG AAGACCCACAACATCGAAGTGGCTCTCAACGAGTTCCCCGATGAGGACCCGGTCGGCCA GGCAGTGGCCCGCGACATGGGCATCGACCTGTACGCCTAA (based on the racemase gene from A. equolifaciens DSM 19450T) BamHI NheI ATCAGGGCAGTACGGATCCTCGTCCGGGGATGCCGACGCATAAGAAAAGAGAAGGAGGC TAGC (sequence from A. equolifaciens DSM 19450T)

ATGGCGTTCGATACTGAGTACGACCTGGTCGTCGTCGGCGGCGGGGCTTCGGGGAAGTC CGCGGCCCTCGAGGCGGCCCGCGCTGGCAAGAGCGTGGTCATCCTCGAGAAAATGCCCG AGACGGGCGGCCTGTCCATGTTCGCCGAGGGCACTGCTGCCTTCGAGTCGAGCGAGCAG AAGAAGCTGGGGAAGCCGGCCCATCCCGACCGTCATTTCCCCACCAAGCAGGAGGGCTA TGACAAGTTCAACGCGTACAGCCACTATCGCGCAAACTTCGACGTGGTGCGCGCCTTCG TGGACAACTCCGCCGATACCATCGATTTCCTCAAGGATCTGGGCGTGGAGTACAAGACG GTGACCATCGCTGCCTATGACGATCCCAACGAGGTGTGGACCTTCCATCTGCCCGACGG CCTGGGCGCTCGCGTGCAGGAACTACTGCTGTCGGCCGTGGAGCACGCCGGTGTGGACA TCTTCACCGAGACTCCGGCCACCGAGCTTATCATTGAGGACGGCAAGGTTGTGGGTGTG GTCGCCGAGTCCGAGGGCGAGCCCCTGCGCGTGGGCGGCAAGGCCGTGGTTCTGGCCAC GGGCGGCATGGGCTCCAACCCCGAGCTCATCGCGAAGTATTCCTGGTTCGCGCCCAGCG CCTACAACATGAACACCCTCACGCCGCTGCAGAACATGGGCGACGGCCTGATGATGGCC CTGTCCGCTGGCGCCGACGACACCAACATCGTCACCTGCCCCATTCTGGCGGCCGGCGC CCGCGACAAGGCCATGGACTCCCACATCGGCGCCGCTGGCGTGCAGCCGGGTGCCATCT GGATCAACAAGACCGGCCGCCGCTTCGCCAACGAGGGCACCGCCGAGAACATCGGCGAT ATCGGCCCGCTGTACGGCAAGCAGCCCGACGGCATTGTGTGGTCGGTGCTCGACCAGGC CAACATCGACCGTCTTGCTGAGAACGGCTCCGAGATCTCCATCGGCGAGTTCGTCGTGT TCGGCCGCCCGCTCGATCGCCTGCGTATGGAGCTCGATCAGGACGTGGCCGACGGCGTG GCCTTCGCGGCCAACACTCCCGAGGAGCTGGCCGAGAAGATCGGCGTGTCCCCGGAGAA CTTCGCCGCTACCGTGGCTACCTACAACGAGGCGTGCGCTACGGGTGACGACAAGGCGT TCTTTAAGCAGGAGAAGTACCTGCGTCCGCTTTCTACCCCGCCGTTCTATGCTGTGGCC CTCGCCACGGGCACCATGGGCTCCGCCGGTGGTATTCGCATCAACGGCAATATGCAGGT AGTAGACAAGGACTACGAGCCCATTCCCGGCCTGTACGCCGTGGGTCTGGACGCTACGG GCCTGTACGGCGACTCCTACAACATGGAGGTGCCGGGCGCGGCTAACGGCTTCGCTCAC

145 CAPÍTULO 3

ACCTCGGGCCGTATCGCGGGCCGTCATTTCGCAGCCCAGCAGGCCTAA (based on the tdr gene from A. equolifaciens DSM 19450T) BamHI CCCTTAGTGCGCAAGGATCCGCACGGCCGATTTCGCGTCGCGCGGTTTTCGTGAGAGAA

NheI GAGATTCGAGAAAGGAGCTAGC (sequence from A. equolifaciens DSM 19450T)

ATGGCAAACATCCCCAAGCTCGTTGGCGCTCCCGATTTCGGTAAGCTCGTCGCTCCGGA GGAGTCTCTGGGCAAGCGATTGGAAGGTAAGCGCGTGTTGCTGACCGGCACCACCAAGG GCGTTGGCCGCGTGACCCAGGAGTTGCTGTGCGCTCAGGGAGCCTTCGTGTGCGGCTCC GGACGCTCCAAGGGCGTGGCTGCCGAGGTGGCCAACGAGTTGGTGGCCAAGGGCTACAA GGCCGCGGGTTTCGACGTGGATCTTTCCGACTACGAGGCCGTGAAGAAGTGGGTGGCCG ACTGCGCCGAGCTCATGGGTGGTATTGATATCGTCATCAACAACGCGTCGCACCCGGGC ATGGCTCCCTTCGGCGAGATGACCCCCGACATCTGGGCTTACGGCATCCAGAACGAGCT CGATCTGGTGTACAACGTGTGCAACTGCGCGTGGCCCTATCTGATCGAGGCCGGTGGCG GCTCCATCATCATCACCTCCTCCACCGTGGGCCTGCAGGGTTCCAACTCCCCCCAGGCC TGCCATGCGGCCGCCAAGGGCGCATGCCTGGCTCTGGCCCGTCAGCTCGCTGCCGAGGG CGGTCCCTTCGGCATCCGCTGCAATTCCATCACCCCCGGTCTGGTGTGGACCGAGGCCA TGTCCAACATCCCCAAGGAGATGGCCCAGGGCCTCATTGCAGCCCAGACCACCCAGCAG GCTATCGATCCGCTCGATATCGCTTACGCCTATCTGTTCCTGGCCTCTGACGAGTCCCG TTTCATCACGGCGGTCAACCTGCCGGTTGACGGCGGCTGCGCTGGTGCGGTAACCGGTG GCATGCAGGGCGAAATCGCTTAA (based on the ddr gene from A. equolifaciens DSM 19450T)

BamHI NheI TTAGGATCCTCGAGCATCTTATCGACTCTTATCATCGTATTAATCAGAAGGGATTGCTA GC (sequence from A. equolifaciens DSM 19450T)

ATGAAGAAGAACCAGCATTTCCCGAAGCTGTTCGAACGCGGCTATATCGCCGGCCTCGA GATCAAGAACCGCATCGTGCGACAGCCCATGGGCACCGAGTTGGGCAATCCCGATGGTT CTCCCAGCTGGGCCACGGTGAAGGCCTATGCCGAGGCAGCCGACGGTGGCGCGGGCATC GTGTATATGGATAACGCCGGTGTGACCCAGTTCCATCATGTGGGCCTGTCCATCGCCAG CGATCCCTACATCGGCCCGATGTCCATCTTGGCCAAGACGCTGAAGCACCACGGTGCGG TGCCCGGCCTGCAGATCGTGCATCCCGGCCGCGACGCCGCCTTCGTGGCAGGCGACGAT CTCATCTCGTCGTCGCGCGTTATGTGGGAGCCTTGGTACGAGAACGGTGGCGGTGTGCC GCGCGAGTTGACCATCGAGGAGATTCACGAGTTCGTGGAGGCCTTCGGCGATGCGGCCG AGCGCGGTCAGCGCGCCGGTTTTGAGATCATCGATGTGCATTCCGCTTGCGGCGTGCTG CTCTCCAACTTCCTGTCGCCGCTCAACAACACCCGTACCGACATGTACGGCGGCTCGCT GCATAATCGCATGCGCTTCCTGATGGAGGTCATTCGCAACATTAAGCAGAAGACCTCGG TGCCGCTGTCCATTCGTCTGTCCGGTTGCGATTTCGAGCCCGGCGGCATCACCATCGAG GAGACCATCGAGGTGGCCAAGGCCTGCGAGCGTATGGGCGCCGACGTCATCAATATCAC CTGGGGCAGTCATGCCGAGGTAGTGAACGCTGCCGGTCTGCTGTCTCCCCACGGCGCAA ACCATGTGGATATGGCCAAGCGCATTAAGGATGCGGTGAGCATTCCGGTCATGTTATGC GGCGGCATCTACACTCCCGAGATTGGCGAGCAGCTGCTGGAAGACGGCGTGTGCGACTA CGTGGGCATCGGCAAGCCGGCTCTGGCCGATCCGTTCTGGGCCAAGAAGGCCGAGGAGG GCCGCTCGGGCGACATTCGCCCCTGCATCGGCTGCGGTGTGGGTTGCCACGACCGCGGT ATGCTGTCGGGCGGTATGGTGCAGTGCGCCGTTAATCCCACACTGTACCAGTTCGATCG CGAGTACTTCCCGAAGACCGATCGTCCGAAAAAGGTAGCCATTATCGGTGCTGGCCCTG CGGGCAGCACTGCGGCTCTGACCGCTGCCGAGTGCGGTCACGACGTGACCCTGTTCGAG

146 CAPÍTULO 3

GGCCGCGAGGTGGGCGGCGTGCTGAAGGAGGCCTCGGTGCCGGTGTACAAGGAGGACCT GGGCCGTCTGGGCAAGTACTACCAGCGCCAGATCGCCAAGTCCAACGTCAAGCTGGTTG AGGAGAATGCCACGCCGGAGACCATCGCTACGGGCGATTTCGACGCTGTGATCGTTGCC ACGGGCGGCAAGGTGCGCGAGTTGAACCTGCCTGGTTTGGATTCCGACAACGTGATTTA CGCCATGGATCTGATGAAGCAGGGGTGTCAGCTGGATGCCGACAAGGTGGTCGTTGTGG GCGGCGGCATCGTGGGTGCCGAGGCGGCTCTCATTCTGGCCGAAGACTTCGGCAAGGAC GTCACCATCACCACGCGCCAGGACAACTTCTTTGTGCCCGGCGTGATGGGCATCGCCTA CATGACACGTCTGGCCATGGCCGGCGTGAAGACGAAGACCCGCGCCAACCTGGTTGAGG TGAAGGACGGCAAGCCGGTGTTCTCCACGATGAATGGTCTGGAGATGATGGATGTGGAT GCCGTGGTGGTGTCGCCCGGCTTCCTGCCCACCAGCCAGATGCGCGACGACATCGAGCA GATTGCCGATGTGGACACCTACGTGATCGGCGACGCCAAGGCCCCGCGTCTGGTGATGG ACGCCGTGCACGAGGGCTACAAGACGGCCATCAACCTGTAA (based on the dzr gene from A. equolifaciens DSM 19450T)

BamHI GGATCCAGAAGGGGTGCCCGCATATTTGGGGCTCCCCTTCTGGAATAAGAACCTTGCTT A HindIII SalI GGCAGCTGACTTCACGACTAGGTGAAGTCAGCTGCCTTTTGCTTTTAAGCTTAGTCGAC CTTG (sequence from A. equolifaciens DSM 19450T)

ggcgtaatcatggtcatagc (sequence from pUC57)

Supplementary Figure 1.- Origin and features of the synthetic DNA designed containing the racemase, and tdr, ddr, and dzr equol genes from Adlercreutzia equolifaciens DSM 19450T. Nucleotides identical to those flanking the multi-cloning site of pUC57 are in lower case letter. The intergenic sequences are those originally present in A. equolifaciens. Start and stop codons and putative ribosome binding sites (RBS) are coloured in pale blue, yellow, and pink, respectively. Some restriction enzyme sites were removed, while all those displayed were added during synthesis.

147

DISCUSIÓN DISCUSSION

Discusión

DISCUSIÓN GENERAL

En los últimos años, en las poblaciones occidentales, se ha desarrollado un creciente interés por el consumo de soja y sus productos derivados, así como por la ingesta de concentrados de isoflavonas. El consumo de soja y sus derivados se ha asociado epidemiológicamente con numerosos beneficios para la salud humana, incluyendo una reducción de la incidencia de diversas enfermedades degenerativas, cardiovasculares y algunos tipos de cáncer (Zaheer y Akhtar, 2017). Los concentrados de isoflavonas, por su parte, se emplean mayoritariamente para el tratamiento de la sintomatología asociada a la menopausia (Smeriglio et al., 2019). Aunque la mecanística del modo de actuación de las isoflavonas no se conoce con certeza, estas presentan gran actividad estrogénica (Yuan et al., 2007), elevada capacidad antioxidante (Choi y Kim, 2014) y son capaces de inhibir algunos enzimas (Crozier et al., 2009), actividades que por separado o combinadas pudieran mediar los beneficios sobre la salud.

En las plantas, las isoflavonas se encuentran unidas a residuos de azúcar en forma de glucósidos. En la soja, principal fuente de isoflavonas, los glucósidos mayoritarios son daidzina, genistina y glicitina. Tras su consumo, mediante la acción de enzimas tisulares y de β-glucosidasas bacterianas, las isoflavonas se desglicosilan (proceso denominado también “activación de las isoflavonas”) dando lugar a las correspondientes agliconas: daidzeína, genisteína y gliciteína (Islam et al., 2015). Estas son más biodisponibles y, en general, tienen mayor actividad biológica. Las agliconas pueden metabolizarse en el intestino, fundamentalmente en el colon, por medio de la acción de enzimas de la microbiota intestinal en metabolitos con mayores actividades fisiológicas (como el equol) o en metabolitos inactivos (como el O-DMA). El equol, derivado de la daidzeína, es el metabolito de las isoflavonas con mayor actividad biológica y al que, por tanto, se le supone un papel primordial en los beneficios para la salud (Franke et al., 2014). Sin embargo, solo entre el 25 y el 35-50% de los individuos de una población (con gran diferencia entre comunidades humanas; Bolca et al., 2007; Peeters et al., 2007), son capaces de producirlo. Estos sujetos poseen en su microbiota intestinal microorganismos capaces de transformar la daidzeína en equol y, por tanto, serían estos los que obtengan mayores beneficios del consumo de isoflavonas (North American Menopause Society, 2011). Si bien en los últimos años se ha incrementado el conocimiento acerca del metabolismo de las isoflavonas y la producción de equol, hay muchas lagunas de

148 Discusión conocimiento sobre los microorganismos que intervienen en el proceso, las rutas metabólicas implicadas y las vías a través de las cuales llevan a cabo sus efectos.

En este contexto, en esta Tesis Doctoral nos hemos propuesto diversos objetivos relacionados con las isoflavonas y su interacción con las poblaciones microbianas intestinales; aspectos en los que pensamos que aún es necesaria mucha información básica. En particular, es esencial disponer de métodos de identificación, cuantificación y monitorización de las poblaciones bacterianas intestinales implicadas en el metabolismo de las isoflavonas y en la producción de equol. Son de interés también las posibles interrelaciones entre las isoflavonas y los constituyentes de la microbiota, ya que por un lado las isoflavonas se metabolizan por biotipos específicos de la microbiota, y por otro las poblaciones intestinales en su conjunto pueden verse afectadas (estimuladas o inhibidas) por estas. En un último apartado, hemos estudiado el efecto de la dieta en la síntesis de equol y se iniciaron trabajos biotecnológicos con el objetivo de producir este compuesto en hospedadores heterólogos. El conocimiento adquirido podría conducir, en último término, a maximizar la producción de equol endógeno y a implementar estrategias destinadas a extender los beneficios del equol a la población en general, con independencia de los taxones presentes en su microbiota.

DETECCIÓN Y CUANTIFICACIÓN DE POBLACIONES PRODUCTORAS DE EQUOL

La mayor parte de las bacterias productoras de equol de origen intestinal (Mayo et al., 2019) se engloban en la actualidad en la familia Eggerthellaceae (Salam et al., 2020). Estas son microorganismos anaerobios estrictos, con grandes requerimientos nutricionales y de crecimiento lento, lo que dificulta su aislamiento en los medios de cultivo convencionales (Browne et al., 2016). Además, son miembros de las poblaciones minoritarias del TGI (Clavel et al., 2014). Todos estos factores complican su purificación de otros biotipos de la microbiota que se encuentran en mayor número o que son capaces de multiplicarse en cultivo de forma mucho más rápida (Rajilić-Stojanović y de Vos, 2014.). Por tanto, la utilización de técnicas independientes de cultivo es una alternativa adecuada para su detección, identificación y cuantificación específicas.

Puesto que la mayoría de los microorganismos descritos como productores de equol pertenecen a la familia Eggerthellaceae, la detección por qPCR de estos se ha realizado hasta el momento mediante la amplificación específica del gen que codifica el ARNr16S de los miembros de esta familia (o de la familia Coriobacteriaceae en la que estaban incluidas tradicionalmente) (Harmsen et al., 2000) o de los géneros Eggerthella o Slackia (Cho et al.,

149 Discusión

2016). Se ha utilizado también la amplificación de secuencias específicas de cepas productoras, como Slackia sp. NATTS (Tsuji et al., 2010; Sugiyama et al., 2014). Sin embargo, en estos momentos, estas amplificaciones dependen de un limitado conocimiento de la microbiota del TGI, así como de los microorganismos productores. De hecho, en la actualidad, no se sabe siquiera si el carácter productor de equol es una característica específica de familia, especie o cepa (Clavel y Mapesa, 2013). Otras limitaciones incluyen la variación en el número de copias del gen que codifica el ARNr 16S en las distintas especies, lo que complica la interpretación de los resultados. Para la síntesis de equol se requiere la actividad de, al menos, los productos de tres genes que codifican reductasas: dzr, que codifica la daidzeína reductasa; ddr, que codifica la dihidrodaidzeína reductasa; y tdr, que codifica la tetrahidrodaidzeína reductasa (Shimada et al., 2010; 2011; Schröder et al., 2013). Estos genes se encuentran en copia única en las bacterias productoras y están emparentados filogenéticamente, lo que permite el diseño de oligonucleótidos “universales” para su amplificación específica.

Basados en las secuencias depositadas en las bases de datos públicas, en este trabajo desarrollamos un método de qPCR altamente específico, sensible y fiable en el que se utilizaron cebadores oligonucleotídicos para amplificar los genes ddr y tdr; la menor homología de los distintos genes dzr no permitió diseñar cebadores universales. Asumiendo un tamaño genómico por célula de ≈3.0 fg de ADN (Rodríguez-Lázaro et al., 2004), estos oligonucleótidos permiten la detección y cuantificación de poblaciones productoras de equol en muestras de heces o cultivos fecales con un límite de detección de alrededor de 102 ufc por gramo o mililitro de muestra. La aplicación de esta metodología mostró que, cuando estaban presentes, los genes tdr y ddr se detectaban en números de copia equivalentes de entre 104 y 105 ufc g-1 o mL-1. Cantidades que se correlacionan bien con las reportadas en trabajos anteriores (Sugiyama et al., 2014; Cho et al., 2016). Como era de esperar, el gen tdr se detectó en todas las muestras de heces procedentes de mujeres productoras de equol (W3, W8, W18). Sin embargo, el gen ddr amplificó sólo en dos de ellas (W3, W8). Estos resultados sugieren la presencia de genes (y quizá bacterias portadoras) distintos a los descritos hasta el momento. De forma sorprendente, se detectaron también amplificaciones positivas para tdr y ddr en muestras de heces y cultivos fecales de dos mujeres con fenotipo no productor de equol. Resultados similares han sido descritos después por otros autores (Braune y Blaut, 2018; Iino et al., 2019). Estas amplificaciones podrían deberse a la presencia de genes no funcionales o a la presencia de genes ortólogos con la suficiente identidad nucleotídica como para amplificar pero que, en la actualidad, codifican enzimas que no poseen actividad sobre la daidzeína y

150 Discusión sus derivados. Alternativamente, y en combinación con los resultados obtenidos en el Capítulo II, Artículo IV de esta Tesis, los genes pudieran estar completos y la falta de funcionalidad podría deberse a deleciones en otros genes del operón esenciales para la síntesis de equol. Los resultados de amplificación mostraron también que el consumo de isoflavonas no incrementaba los niveles de microorganismos productores de equol en heces, al menos durante el tiempo controlado (seis meses), en contra de lo que han sugerido otros autores (Nakatsu et al., 2014; Guadamuro et al., 2017; Iino et al., 2019). Tampoco se detectó incremento de los organismos productores en cultivos fecales en medio de cultivo con isoflavonas en un modelo de intestino artificial (Capítulo III, Artículo VI).

Dado el escaso conocimiento sobre los microorganismos productores de equol, junto con los resultados obtenidos mediante qPCR en esta tesis (Artículo I) y en otros de trabajos previos del grupo (Guadamuro et al., 2015; Guadamuro et al., 2017), es fácil pensar que los niveles reales de productores podrían estar subestimándose. Es decir, que, por algún motivo, no estamos contando todos los biotipos capaces de sintetizar equol. De forma reciente, se han utilizado técnicas de secuenciación metagenómica dirigida, basada en la amplificación de una región del gen que codifica el ADNr 16S, y no dirigida (“shotgun”) para la monitorización de poblaciones bacterianas involucradas en el metabolismo de las isoflavonas en muestras fecales (Guadamuro et al., 2019; Iino et al., 2019; Zheng et al., 2019). Mediante estas técnicas, se han descrito niveles superiores de microorganismos pertenecientes a taxones relacionados con el metabolismo de las isoflavonas como los géneros Eggerthella o Collinsella (Guadamuro et al., 2019), o de especies productoras de equol como Asaccharobacter celatus y Slackia isoflavoniconvertens, en muestras fecales de individuos con fenotipo productor de equol (Iino et al., 2019). Sin embargo, al igual que ocurría con los resultados de la qPCR, los taxones relacionados con la síntesis de equol se detectan tanto en muestras de individuos productores como de no productores (Iino et al., 2019). Las limitaciones impuestas por las técnicas de amplificación (Jian et al., 2020) quizá podrían superarse mediante el empleo de técnicas moleculares con gran profundidad de análisis y que no estén basadas en la PCR. Estas técnicas podrían ser útiles para relacionar el fenotipo productor de equol con la composición y funcionalidad de la microbiota intestinal.

Tratando de profundizar en estas discrepancias, en este trabajo se utilizó la secuenciación metagenómica shotgun del ADN microbiano total de muestras fecales de mujeres productoras y no productoras. Como se esperaba, la composición bacteriana

151 Discusión resultó diferente en cada uno de los individuos, aunque, en términos de diversidad taxonómica -un parámetro muy relacionado con la capacidad metabólica (Le Chatelier et al., 2013)-, los resultados fueron similares entre las muestras de mujeres productoras y no productoras de equol. A pesar de eso, la diversidad metabólica deducida fue mayor en las muestras de mujeres productoras de equol. Las poblaciones mayoritarias pertenecían al filo Firmicutes, seguidas por otras de los filos Actinobacteria y Bacteroidetes. Representantes de la clase Coriobacteriia, incluyendo especies productoras de equol, se detectaron en muestras de mujeres productoras y no productoras con una abundancia relativa en ambos casos de entre el 0.9% y el 3.3%. Las lecturas más abundantes correspondían a las especies A. equolifaciens, Eggerthella lenta y Gordonibacter pamelaeae. Tal y como se había observado en los resultados de qPCR, también mediante shotgun se detectaron taxones productores de equol en muestras de mujeres productoras y no productoras. Utilizando esta misma técnica (Zheng et al., 2019) y técnicas de metagenómica filogenética (Iino et al., 2019) ya se habían reportado resultados similares con anterioridad. Algunos autores interpretan estos resultados sugiriendo que la producción de equol no depende de la cantidad de microorganismos productores sino de la “calidad” de los mismos (Yoshikata et al., 2019). En esta calidad, como se verá a continuación, podríamos incluir la presencia de operones no funcionales en algunos o en todos los taxones “productores” presentes en ciertos individuos. De estos resultados se deduce que la caracterización del fenotipo productor de equol no puede basarse exclusivamente en un análisis taxonómico. La aproximación metagenómica permitió caracterizar también la funcionalidad de los genes presentes en las muestras fecales analizadas. En las muestras de mujeres con fenotipo productor, se observó una mayor diversidad funcional y un mayor número de secuencias que codifican enzimas relacionadas con el metabolismo de carbohidratos y la síntesis y degradación de AGCC. Sin embargo, no se identificaron genes o rutas metabólicas relacionadas con el metabolismo de las isoflavonas, excepto por unas pocas secuencias con homología con oxidorreductasas dependientes de FAD y glutamato sintasas. En cuanto a genes involucrados en la producción de equol, se detectó en una sola muestra una secuencia homóloga al gen dzr de A. equolifaciens DSM19450T. En definitiva, la tecnología de secuenciación shotgun utilizada no tuvo la suficiente profundidad de análisis y/o cobertura para permitir la detección (y caracterización) de genes involucrados en la producción de equol.

152 Discusión

EFECTO DE LAS ISOFLAVONAS SOBRE LA MICROBIOTA INTESTINAL

Los efectos beneficiosos sobre la salud humana asociados al consumo de soja y productos derivados se atribuyen fundamentalmente a su contenido en isoflavonas. Estas, como se ha comentado, podrían ejercer sus efectos mediante su actividad estrogénica, antioxidante o la inhibición de ciertos enzimas (Zaheer y Akhtar, 2017). Sin embargo, también es posible que las isoflavonas modulen la composición y actividad de las poblaciones intestinales, y que esta modulación participe de alguna forma (p. ej., con el aumento de poblaciones o metabolitos beneficiosos) en los efectos saludables. Aunque escasos y con resultados poco concluyentes, dada presumiblemente la enorme variabilidad interindividual de la microbiota intestinal (Lozupone et al., 2012), existen estudios que evalúan los efectos de las isoflavonas sobre microorganismos intestinales y analizan los cambios en la composición de las poblaciones bacterianas durante el consumo de isoflavonas. Directa o indirectamente, las isoflavonas, podrían alterar la composición microbiana favoreciendo o inhibiendo el crecimiento de determinados grupos bacterianos en el ecosistema intestinal. Así, la presencia de isoflavonas en la dieta se ha relacionado con un aumento de especies de los géneros Bifidobacterium (Nakatsu et al., 2014), Collinsella, Faecalibacterium o de miembros de Clostridium de los clústeres IV and XIVa (Guadamuro et al., 2017). El fenotipo productor de equol también se ha asociado con una abundancia de bacterias reductoras de sulfato (Desulfovibrio sp.) (Bolca et al., 2007), bacterias implicadas en varias enfermedades (Loubinoux et al., 2002).

Efecto de las isoflavonas sobre el crecimiento bacteriano

La actividad antimicrobiana de las isoflavonas frente a microorganismos patógenos resistentes a antibióticos ya se ha evaluado en estudios previos (Verdrengh et al., 2004; Hummelova et al., 2015). También se ha examinado el empleo de otros compuestos fenólicos con capacidad antimicrobiana como aditivos naturales para la preservación de alimentos (Villalobos et al., 2016; Wang et al., 2018; Freitas et al., 2019). Ahondando en el tema, nosotros hemos analizado el efecto antimicrobiano de las isoflavonas frente a un grupo de especies bacterianas representativas del tracto gastrointestinal humano, incluyendo bacterias ácido-lácticas, bifidobacterias y otras especies intestinales de grupos mayoritarios. Los resultados obtenidos mostraron muy poca actividad antimicrobiana para la daidzina, genistina y sus formas agliconas (daidzeína y genisteína), con una concentración inhibitoria mínima (CIM) de 2048 µg mL-1 para todas las cepas ensayadas. En el caso del equol, los resultados fueron más heterogéneos con un valor de CIM que iba desde los 16 µg mL-1 para Bifidobacterium animalis subsp. animalis, la cepa más sensible,

153 Discusión hasta los 2048 µg mL-1 para Escherichia coli y Klebsiella pneumoniae. En general, para las cepas examinadas con anterioridad los resultados concordaban con los de la literatura. Así, se ha reportado una ligera actividad antimicrobiana de isoflavonas como la genisteína frente a las bacterias Gram-negativas E. coli y Helicobacter pylori (Verdrengh et al., 2004), o de la genisteína y la daidzeína sobre E. coli y Pseudomonas aeruginosa (Hummelova et al., 2015; Wang et al., 2018). La ausencia de grupos prenilo e hidroxilo en determinadas posiciones de los anillos de las isoflavonas (presentes en otros polifenoles), explicaría su baja acción antimicrobiana (Mukne et al., 2011). Aunque el efecto de las isoflavonas sobre los microorganismos intestinales depende de las especies, y quizá de las cepas, las bacterias Gram-negativas parecen mostrar una menor inhibición que las Gram-positivas. En este sentido, se ha descrito que la genisteína es capaz de inhibir el crecimiento de Streptococcus pasteurianus, Bacillus cereus y todas las cepas ensayadas de Staphylococcus aureus, incluyendo cepas resistentes a meticilina (MRSA) (Verdrengh et al., 2004). No obstante, hay que puntualizar que en los análisis llevados a cabo en este trabajo se utilizaron concentraciones de isoflavonas muy por encima de los niveles fisiológicos que se alcanzan en el TGI (≈2 µg g-1 de heces; Guadamuro et al., 2015b).

Aunque nuestros resultados apenas mostraban actividad antimicrobiana, en un intento por profundizar en el posible efecto inhibidor de las isoflavonas, se evaluaron también parámetros relacionados con el crecimiento en 10 cepas bacterianas pertenecientes a los grupos más representativos. En el perfil de las curvas de crecimiento no se observaron cambios destacables e, incluso, durante las primeras 8 horas la mayoría de las cepas presentaban el mismo perfil de crecimiento en presencia o no de isoflavonas. Aun así, para algunas cepas se observaron pequeñas alteraciones que apuntaban la idea de que las isoflavonas podrían tener un efecto sobre el desarrollo de determinadas poblaciones. La genisteína, y en menor medida el equol, inhibía el crecimiento de las especies Faecalibacterium prausnitzii, Lactococcus lactis subsp. lactis y Bacteroides fragilis. Por el contrario, el equol parecía promover el crecimiento de E. coli y Serratia marcescens. Resultados similares se obtuvieron sobre la tasa de crecimiento (µ) y la densidad óptica final de los cultivos. En presencia de genisteína y equol, la tasa de crecimiento disminuyó de forma considerable en B. fragilis, L. lactis subsp. lactis y Slackia equolifaciens y aumentaba en Lactobacillus rhamnosus y F. prausnitzii. Este hecho sugiere que, de algún modo, estas cepas pudieran ser capaces de degradar y utilizar estos compuestos como fuente de energía. Durante los cultivos, las formas glicosiladas de las isoflavonas se desconjugan por la acción de glicosil-hidrolasas liberándose moléculas de glucosa que pudieran ser utilizadas por los microorganismos (Islam et al., 2015). Con excepción de este

154 Discusión proceso de desglicosilación, el conocimiento acerca de las rutas metabólicas implicadas en el metabolismo de las isoflavonas por estas especies es desconocido (Franke et al., 2014). En su conjunto, estos resultados invitan a pensar que las isoflavonas y sus metabolitos derivados pudieran modular el crecimiento de algunas poblaciones bacterianas del intestino, lo que podría estar relacionado con su efecto beneficioso sobre la salud.

Biotipos microbianos implicados en la producción de equol

El limitado conocimiento de las bacterias implicadas en la producción de equol, junto con la enorme diversidad bacteriana del TGI (Lozupone et al., 2012), insinúan la posible existencia de biotipos bacterianos pertenecientes a otros taxones que intervengan en el metabolismo de las isoflavonas y en la producción de equol. De hecho, en esta tesis se han detectado reductasas directamente implicadas en la producción equol con una secuencia nucleotídica distinta hasta las ahora descritas (Capítulo I, Artículo I). Además, la detección fallida del gen ddr en una muestra de una mujer productora indica la posible presencia de genes distintos a los que se conocen y utilizaron en el diseño de los oligonucleótidos.

En este contexto, nos propusimos el estudio de la actividad sobre daidzeína y genisteína de bacterias intestinales aisladas a partir de muestras fecales de mujeres productoras de equol, portadoras, por tanto, de microorganismos capaces de metabolizar la daidzeína. Entre los biotipos analizados se detectaron cepas de diversas especies con capacidad para transformar genisteína y daidzeína en dihidrogenisteína o dihidrodaidzeína y O-DMA, respectivamente. No se descarta que algunos aislados conviertan las isoflavonas en metabolitos no identificados (Kim et al., 2009; Lee et al., 2017), ya que el método cromatográfico empleado (Redruello et al., 2015) requiere la utilización de patrones comerciales para llevar a cabo la detección y cuantificación de los analitos; muchos de estos resultan difíciles de adquirir o precisan ser sintetizados. Ninguna de las cepas ensayadas fue capaz de producir equol, ni en cultivo puro ni mixto, descartando la complementariedad de reacciones que permitiesen completar la ruta metabólica conocida de producción de equol (Decroos et al., 2005). En este sentido, la daizeína se puede reducir a dihidroequol por medio de una ruta alternativa al equol y también a través de los intermediarios dihidrodaidzeína y tetrahidrodaidzeína (Kim et al., 2009). Otra posible explicación es la metabolización subsecuente del equol que se va produciendo, algo que en nuestro caso no hemos podido confirmar ni en cultivos de A. equolifaciens (Capítulo III, Artículo VII) ni en cultivos fecales (resultados no publicados). Una mejor caracterización metabólica y enzimática de las cepas productoras de equol permitiría el desarrollo de nuevas aproximaciones basadas en enriquecimientos selectivos

155 Discusión en cultivo. En este sentido, se ha descrito que la suplementación de los medios con sangre, arginina o surfactantes (como Tween 80), favorecen el crecimiento de miembros de la familia Coriobacteriaceae (Clavel et al., 2014). Algunas especies productoras de equol son capaces de resistir a la bilis o a determinados antibióticos que podrían incorporarse a los medios de cultivo para su aislamiento selectivo (Atkinson et al., 2004). El medio GAM suplementado con arginina utilizado en este trabajo está indicado para el crecimiento de poblaciones bacterianas intestinales minoritarias (Lozupone et al., 2012) y ya ha sido empleado en el aislamiento de cepas productoras de equol (Maruo et al., 2008; Minamida et al., 2008; Jin et al., 2010). Con todo, los autores reconocen que la metodología empleada hasta ahora para el aislamiento de microorganismos productores de equol no es muy adecuada, ya que, por el momento, no existe ningún paso mínimamente selectivo.

Análisis genómico de bacterias productoras de equol

Entre las bacterias aisladas en este trabajo resultó sorprendente identificar un aislado de la especie A. equolifaciens que no producía equol a partir de daidzeína. Para profundizar en el motivo, se secuenció su genoma, y el análisis de su secuencia y las proteínas deducidas situaron al aislado IPLA 37004 en una rama muy próxima a las especies A. equolifaciens y A. celatus. De forma reciente, se ha propuesto que estas especies representan el mismo taxón (Salam et al., 2020). Como era de esperar por su fenotipo no productor, en la cepa aislada se encontraban ausentes las 13 ORFs que integran el clúster de producción de equol en A. equolifaciens DSM 19450T. Sin embargo, las regiones que flanquean esta estructura génica mostraban una alta identidad nucleotídica y aminoacídica en las distintas cepas. Una comparación más extensa de los genomas de las especies A. equolifaciens y A. celatus reveló la alternancia de cepas portadoras del clúster del equol con otras en las que no está presente y que, presumiblemente, no producen equol. La gran linealidad y la elevada conservación de las regiones flanqueantes del clúster sugiere que las cepas que carecen de operón han sufrido una deleción de esta región génica. La pérdida génica podría explicarse si el fenotipo productor de equol no ofreciera ninguna ventaja selectiva a estos microorganismos en el ambiente intestinal, de manera que la pérdida metabólica no afectaría a su desarrollo. En todo caso, parece claro que, tal como se ha reportado en este trabajo (Capítulo I, Artículo I y II) y en la literatura (Braune y Blaut, 2018; Iito et al., 2019), en el TGI de muchas personas no productoras de equol existen taxones idénticos a los que se detectan en los individuos productores. La dieta es uno de los factores con mayor influencia sobre la microbiota intestinal humana (Graf et al., 2015; Leeming et al., 2019), y se encuentra bien establecido que la frecuencia de

156 Discusión individuos productores de equol en poblaciones vegetarianas es mayor con respecto a aquellas que no lo son (Setchell y Cole, 2006). Una dieta rica en productos vegetales con gran carga de polifenoles concuerda con la conservación del fenotipo productor de equol en todos los animales estudiados (Schwen et al., 2012). Mientras que el abandono de una ancestral dieta humana (paleodieta) a base de plantas, frutas y tubérculos (Ma et al., 2016) y su sustitución por un mayor consumo de carne y productos animales, pudiera estar favoreciendo la pérdida del operón en las bacterias intestinales productoras. Los taxones no productores podrían, de alguna forma, ser más competitivos en la actualidad que los taxones productores.

MAXIMIZACIÓN DE LA PRODUCCIÓN DE EQUOL

Como se ha señalado anteriormente, es posible que sólo los individuos que alberguen en su interior los microorganismos responsables de la conversión de daidzeína en equol (un 25-50% de los individuos según la población) sean capaces de beneficiarse completamente del consumo de isoflavonas. Por ello, la extensión de los beneficios de este compuesto al conjunto de la sociedad con independencia de los microorganismos presentes en su TGI resulta un objetivo atractivo. Al respecto, profundizar en el conocimiento de los enzimas que participan en su síntesis y los mecanismos de regulación implicados es de suma importancia para el diseño de estrategias que permitan incrementar los niveles de síntesis endógena de equol y/o mejorar la producción biotecnológica a gran escala para su empleo, por ejemplo, en alimentación funcional.

Incremento de la producción endógena de equol

Tal como se expone en los párrafos precedentes, la dieta es uno de los factores con mayor influencia sobre la microbiota intestinal. En este contexto, parece fácil suponer que la dieta es capaz de modular las poblaciones intestinales y en consecuencia, tener un efecto también sobre las productoras de equol. Diversos estudios previos han evaluado la influencia de determinados componentes de la dieta sobre la producción de equol en varios modelos animales y en el hombre. Así, se ha descrito que el consumo de almidón resistente por ratones ovariectomizados (Tousen et al., 2011) promueve la producción de equol a partir de la isoflavona precursora. El mismo efecto parece tener la lactulosa en hembras de cerdo (Zheng et al., 2014) o el xilitol en ratones (Tamura et al., 2013). En el hombre, el consumo de leche y productos lácteos en combinación con daidzeína se correlaciona con un incremento de la producción de equol (Frankenfeld, 2011a). En este

157 Discusión trabajo nos propusimos evaluar la influencia de distintos regímenes alimentarios sobre la producción de equol utilizando un modelo de intestino artificial (TIM-2) (Venema, 2015).

Los resultados de este trabajo, al igual que los reportados en la literatura y arriba referenciados, mostraron una influencia clara de la dieta en la producción de equol. Una dieta con isoflavonas y rica en carbohidratos dobla la producción de equol, respecto de una dieta estándar, mientras que la producción disminuye a la mitad con una dieta rica en proteínas. Este resultado pone de manifiesto que la dieta podría ser un importante factor modulador de la producción endógena de equol. La cantidad de equol aumenta a lo largo de la fermentación de forma que la máxima cantidad se observa a las 72 h, lo que sugiere, como ya habíamos avanzado, la estabilidad del producto final (equol). La formación de O- DMA, también parece fuertemente inhibida con la dieta rica en proteínas. Este compuesto no tiene poder estrogénico, pero bien pudiera tener otras actividades fisiológicas beneficiosas sobre la incidencia o el curso de determinadas enfermedades (Frankenfeld, 2011b). Por su parte, los grupos bacterianos relacionados con la producción de equol (Slackia, Eggerthella) no se ven favorecidos en los cultivos ni por la presencia de isoflavonas ni con las distintas dietas. El mecanismo por el que se incrementa la actividad productora de equol es, por el momento, desconocido.

Regulación transcripcional del clúster implicado en la producción de equol

La síntesis de equol a partir de daidzeína tiene lugar por acción consecutiva de reductasas específicas de origen bacteriano a través de los intermediarios metabólicos dihidrodadizeína y tetrahidrodaidzeína (Kim et al., 2009). Como ya se ha adelantado, los genes que codifican la reductasas (tdr, ddr y dzr) se describieron por primera vez en la cepa de L. garvieae 20-92 (Shimada et al., 2010; 2011). En esta misma cepa se identificó más tarde un cuarto gen con actividad racemasa, situado delante de las reductasas y necesario para una producción de equol eficiente (Shimada et al., 2012). La misma organización genética se encuentra presente también en S. isoflavoniconvertens (Schröder et al., 2013), Slackia sp. NATTS (Tsuji et al., 2012), A. equolifaciens (Toh et al., 2013) y Eggerthella sp YY918 (Kawada et al., 2016). Con el objetivo de profundizar en el estudio de los mecanismos de regulación de la producción de equol, en esta Tesis se llevó a cabo el análisis transcripcional de los genes del clúster de equol en la cepa productora A. equolifaciens DSM 19450T.

Trabajos previos en L. garvieae y S. isoflavoniconvertens (Shimada et al., 2010; Schröder et al., 2013) sugerían que el clúster de biosíntesis de equol estaba integrado por unas 8

158 Discusión pautas abiertas de lecturas (ORFs). Los resultados llevados a cabo en este trabajo revelaron la presencia de al menos 13 genes contiguos, los cuales incrementaban su expresión en presencia de daidzeína. Esto sugiere que todos ellos, de un modo u otro, están relacionados con la producción de equol. Como era de esperar, tres de ellos se correspondían con las reductasas imprescindibles y otro más con la racemasa. Otros dos genes codificaban para subunidades de flavoproteínas cuya función pudiera ser la de dotar de poder reductor, imprescindible al menos para el enzima daidzeín reductasa en la reducción de la daidzeína (Kawada et al., 2018). Nada se sabe sobre la posible función del resto de genes que conforman el clúster. Se especula, por ejemplo, que pudieran codificar diversos cofactores que se unen a las reductasas, tal como se ha descrito para la daidzeín reductasa de Eggerthella (Kawada et al., 2018). El análisis transcriptómico del clúster sugiere que los genes se encuentran organizados en forma de un operón. La expresión génica de los genes, incluyendo tdr, ddr y dzr, aumentaba de manera proporcional a la concentración de daidzeína en el medio entre 0.5 y 4.0 unidades logarítmicas. Además, en el operón se distinguieron cuatro patrones de expresión transcripcional, relacionados in silico con la presencia de cuatro posibles secuencias terminadoras, algunas ya reportadas con anterioridad en S. isoflavoniconvertens (Schröder et al., 2013). Sin embargo, el análisis transcripcional de las regiones intergénicas reveló que el operón completo se transcribía como una única molécula de ARN mensajero, sugiriendo la presencia de mecanismos postranscripcionales que regulen la expresión diferencial de genes (Conway et al., 2014).

Clonación del clúster de producción de equol en E. coli y BAL

En su gran mayoría, las bacterias descritas como productoras de equol pertenecen a la Clase Coriobacteriia y se caracterizan por ser microorganismos anaerobios estrictos, muy sensibles a la presencia de oxígeno en el medio y con grandes requerimientos nutricionales (Salam et al., 2020), motivos por los que son difíciles de cultivar. Como se ha señalado anteriormente, el equol es el metabolito de las isoflavonas con mayor actividad biológica y al que se le supone un papel primordial en los beneficios para la salud. Disponer de grandes cantidades de equol posibilitaría llevar a cabo las intervenciones experimentales necesarias para determinar su papel en la salud humana. Si fuera positivo, el equol purificado podría utilizarse directamente para extender sus beneficios a todos los individuos, con independencia de la composición de su microbiota.

En este contexto, el objetivo principal de este trabajo consistió en transferir la maquinaria genética encargada de la producción de equol desde un organismo productor a otros más manejables, con el fin de disponer de sistemas bacterianos simples para una

159 Discusión síntesis barata y a gran escala del compuesto. Estudios de este tipo ya se encuentran reportados en la literatura. Así, en E. coli ya se han clonado y expresado los genes que codifican las tres reductasas de L. garvieae 20-92 (Shimada et al., 2010; 2011) y los genes equivalentes de Slackia sp NATTS, S. isoflavoniconvertens y Eggerthella sp. YY7918 (Tsuji et al., 2012b; Schröder et al., 2013; Kawada et al., 2016; Lee et al., 2016). En esta Tesis se determinó clonar la maquinaria genética de producción de equol de A. equolifaciens DSM 19450T, cepa que habíamos utilizado como modelo en los trabajos precedentes. Para ello, a partir de la secuencia genómica de A. equolifaciens DSM 19450T se diseñó un fragmento de ADN sintético portador de los tres genes que codifican las reductasas y el gen de la racemasa, precedidos en el inicio por un promotor fuerte y constitutivo de L. lactis.

La construcción se introdujo en E. coli por medio del vector de clonación pUC57, y en las BAL Lactobacillus casei y Lactococcus lactis utilizando el vector pIL252. La funcionalidad de los clones para la producción de equol se comprobó mediante UHPLC con el método cromatográfico que hemos venido utilizando a lo largo de la Tesis (Redruello et al., 2015). En cultivos de E. coli portadores de la construcción pUC57-equol, aproximadamente el 90% de la daidzeína se convierte en equol mientras que en presencia de dihidrodadizeína se produce una transformación parcial y sólo el 15% se convierte en equol. Aunque con eficiencias distintas según el precursor, estos resultados demuestran que los genes sintéticos son funcionales en esta bacteria hospedadora. También resultó funcional en E. coli la construcción pIL252-pUC57-equol, con la que se convertía en equol aproximadamente el 5% de la dadizeína y de la dihidrodadizeína. Estos resultados no resultan sorprendentes, ya que se ha visto que la expresión de los genes de producción de equol de distintas especies y cepas en E coli es muy variable (Schröder et al., 2013; Kawada et al., 2016; Lee et al., 2016). La menor producción de equol de los clones recombinantes portadores de pIL252 podría deberse a un aumento del tamaño de la construcción o a una disminución de la estabilidad estructural del clon. Esto último se apoya en la observación de que mutaciones del gen ddr y en otros genes constitutivos se ha visto que contribuyen a la estabilidad de los clones y, en consecuencia, a una mayor producción de equol (Lee et al., 2016).

En el caso de las BAL recombinantes portadoras de la construcción pIL252-pUC57- equol, sólo se observó producción en L. casei y únicamente a partir de dihidrodaizeína. Estos resultados indican que la dihidrodaidzeín reductasa y la tetrahidrodadizein reductasa se expresan y son funcionales. En L. lactis, sin embargo, las cepas recombinantes no produjeron equol a partir de ninguno de los precursores utilizados. La presencia de

160 Discusión mutaciones inactivadoras en las secuencias de los genes se descartó, puesto que el ADN procedente de las BAL se retransformó de nuevo en E. coli, y las células transformantes producían de nuevo equol a partir de la daidzeína. A priori, la producción de equol en BAL y en concreto en Lactococcus sp. no debería suponer mayor problema puesto que el clúster de producción de equol de L. garvieae ha sido probablemente adquirido por transferencia horizontal desde cepas de la familia Eggerthellaceae (Shimada et al., 2010; 2011). Sin embargo, las BAL puede que no reconozcan determinadas señales nativas (promotor, sitio de unión al ribosoma) de alguno de los genes de A. equolifaciens. La expresión de estos quizá requiera el reemplazo de las señales de expresión originales por otras específicas de las BAL.

A pesar de los numerosos controles legales del uso de microorganismos recombinantes previstos en la legislación europea, las cepas recombinantes de E. coli podrían emplearse para la producción de equol con el que desarrollar aditivos o alimentos funcionales enriquecidos en este compuesto. La producción de equol en BAL, por su parte, posibilitaría su formación concomitantemente con la fermentación de bebidas de soja.

En conclusión, aunque son necesarios más estudios para resolver muchas de las cuestiones que quedan pendientes de las relaciones de las isoflavonas con la microbiota, de los microorganismos de origen intestinal capaces de producir equol, de los mecanismos que dirigen esta producción en las condiciones fisiológicas del intestino humano y del control de la producción en las bacterias productoras, pensamos que los resultados de esta Tesis constituyen una pequeña contribución para dilucidar las complejas relaciones isoflavonas-microbiota, apuntan a una posible pérdida del fenotipo productor en las bacterias del intestino humano, documentan un incremento de la producción con una dieta rica en carbohidratos y sientan las bases para profundizar en una futura maximización de la expresión de la maquinaria metabólica de producción de equol en hospedadores heterólogos.

161

CONCLUSIONES CONCLUSIONS

Conclusiones

CONCLUSIONES

PRIMERA.- Se ha desarrollado y validado un método de qPCR basado en la amplificación de secuencias conservadas de dos genes (tdr y ddr) que codifican reductasas involucradas en la síntesis de equol. El método se utilizó para la detección y cuantificación de microorganismos productores de este compuesto en muestras de heces o cultivos fecales. Cuando están presentes, los genes tdr y ddr mostraron un número de copias similar de entre 104 y 105 por gramo o mililitro de muestra. De forma sorprendente, estos genes estaban presentes también en muestras de algunas mujeres no productoras de equol. El consumo de isoflavonas o la presencia de éstas en los medios de cultivo no incrementa el número de copias. El conocimiento limitado de los microorganismos productores de equol y las rutas metabólicas implicadas, así como una posible pérdida de esta función metabólica en determinados taxones, como se verá más adelante, podría estar detrás de gran parte de las incongruencias detectadas.

SEGUNDA.- La secuenciación metagenómica total no dirigida (“shotgun”) de ADN total de muestras de heces permitió la detección de taxones de microorganismos productores de equol; estos aparecieron en números similares en muestras de heces de mujeres productoras y no productoras. Sin embargo, la tecnología y profundidad de secuenciación utilizadas no fueron suficiente para detectar y cuantificar genes involucrados en su síntesis. En las muestras analizadas, solo se detectó en una sola muestra una única secuencia homóloga al gen dzr de Adlercreutzia equolifaciens DSM19450T.

TERCERA.- Las isoflavonas y sus principales metabolitos derivados no tienen un gran efecto antimicrobiano, aunque pueden influenciar de forma selectiva en parámetros relacionados con el crecimiento bacteriano de algunas especies. Así, la genisteína y el equol favorecen la tasa de crecimiento de Lactobacillus rhamnosus y Faecalibacterium prausnitzii y mientras que la genisteína reduce este mismo parámetro en cepas de, Slackia equolifaciens, Lactococcus lactis y Bacteroides fragilis. Estos pequeños efectos pudieran ser suficientes para modular las poblaciones bacterianas de la microbiota intestinal.

CUARTA.- Se ha analizado el metabolismo de las isoflavonas de la soja daidzeína y genisteína por bacterias intestinales pertenecientes a cuatro filos, 10 familias y 21 especies. Cepas de taxones diferentes produjeron dihidrogenisteína o dihidrodaidzeína y O-desmetilangolensina (O-DMA) a partir, respectivamente, de la genisteína y la daidzeína. A pesar de partir de un procedimiento selectivo por qPCR utilizando como blanco el gen

162 Conclusiones tdr, no se detectaron cultivos puros ni mezclas capaces de producir equol a partir de la daidzeína.

QUINTA.- Se aisló, identificó y secuenció el genoma de A. equolifaciens IPLA 37004, cepa que, a pesar de pertenecer a una especie productora de equol, no producía equol de daidzeína. En consonancia, el análisis genómico de IPLA 37004 reveló la ausencia de las 13 ORFs que conforman el clúster del equol en la cepa A. equolifaciens DSM19450T. La ausencia de este clúster y la conservación de las regiones adyacentes en varias cepas de esta especie y de la especie relacionada Asaccharobacter celatus sugiere que, en el ambiente intestinal humano, el fenotipo productor de equol no provee una ventaja selectiva.

SEXTA.- La dieta, uno de los factores moduladores de la microbiota intestinal, influye también en la producción de equol. En cultivos fecales realizados en un modelo dinámico de colon proximal humano (TIM-2), una dieta rica en carbohidratos incrementó al doble la producción de equol en comparación con una dieta balanceada, mientras que una dieta con un alto contenido de proteínas la redujo a la mitad. Estas alteraciones en la producción de equol, sin embargo, no llevaban aparejados cambios significativos en los niveles de los microorganismos productores.

SÉPTIMA.- El análisis transcripcional de las ORFs del clúster de equol en la bacteria productora A. equolifaciens DSM19450T, analizado mediante técnicas de RT-PCR y RT- qPCR, permitió determinar que constituía un solo operón integrado por 13 genes contiguos, entre los que se encontraban los genes tdr, ddr y dzr. La presencia de daidzeína, la isoflavona precursora de equol, induce la expresión de todos los genes del operón, detectándose cuatro patrones de expresión diferencial; estos coinciden con la presencia de posibles secuencias promotoras y terminadoras.

OCTAVA.- Basados en las secuencias de A. equolifaciens DSM19450T, se han diseñado y sintetizado cuatro genes involucrados en la producción de equol (racemasa, tdr, ddr y dzr). Estos se clonaron en cepas modelo de Escherichia coli, Lactobacillus casei y L. lactis, bajo el control de un promotor de esta última especie. Las cepas recombinantes de E. coli produjeron equol a partir de la daidzeína, mientras que las de L. casei solo lo producían a partir del intermediario dihidrodaidzeína y las de L. lactis no produjeron equol. La producción biotecnológica de equol en bacterias heterólogas fáciles de cultivar posibilitaría una síntesis barata y segura a gran escala de este compuesto. La síntesis de equol en bacterias ácido-lácticas requerirá de más ensayos, utilizando, por ejemplo, señales específicas de transcripción y traducción.

163 Conclusions

CONCLUSIONS

FIRST.- A real-time quantitative PCR method based on the amplification of conserved sequences of two reductase-encoding genes (tdr and ddr) involved in the equol biosynthesis for the detection and quantification of equol-producing microorganisms in faecal samples or their derived slurry cultures was developed and validated. When present, the two genes were detected in similar copy numbers ranging between 104 and 105 per gram or millilitre of sample. Surprisingly, the genes were scored in samples from equol producing and equol non-producing women. Isoflavone consumption or faecal cultures in the presence of isoflavones did not significantly increase the copy number of equol-related genes. The limited knowledge of the microorganisms and metabolic pathways involved in equol production, together with a likely loss of function in some equol-producing taxa, see below, may account for inconsistency in the results.

SECOND.- The current shotgun metagenomic sequencing technology and the depth of sequencing (coverage) of total DNA from stool samples allowed the detection of microbial taxa involved in equol production; these appear in similar numbers in stool samples from both equol producing and non-producing women. However, this technique was not powerful enough to detect and quantify genes involved in equol biosynthesis. In the analysed samples, only one sequence homologous to the dzr gene of Adlercreutzia equolifaciens DSM19450T was identified in a single sample.

THIRD.- Isoflavones and their derived metabolites did not show significant antimicrobial activity in intestinal bacteria, although these compounds can selectively modify growth- related parameters to some species. As such, both genistein and equol increased the growth rate of Lactobacillus rhamnosus and Faecalibacterium prausnitzii, while genistein reduced this parameter in Slackia equolifaciens, Lactococcus lactis, and Bacteroides fragilis. These effects might be sufficient for isoflavone consumption to modulate the development of some bacterial populations of the gut microbiota.

FOURTH.- Analysis of the metabolites from daidzein and genistein isoflavones produced by intestinal biotypes belonging to four phyla, 10 families, and 21 species, proved that strains of different taxa generate dihydrogenistein or dihydrodaidzein and/or O- desmethylangolensin (O-DMA) from their respective precursor. Despite using a selective qPCR procedure for the selective detection of the tdr gene, none of the isolates was

164 Conclusions reported to produce equol as either pure cultures or in combinations of strains from different species.

FIFTH.- An A. equolifaciens strain was isolated and identified, but it did not produce equol from daidzein. Genome sequencing and analysis of A. equolifaciens IPLA 37004 revealed the lack of the complete equol operon present in A. equolifaciens DSM19450T, which consisted in a gene cluster of 13 ORFs. The absence of this cluster and the highly conserved linearity in both upstream and downstream sequences in the DNA of some strains of A. equolifaciens and those of the related species Asaccharobacter celatus, suggests that the equol-producing phenotype might not currently provide a selective advantage in the human gut ecosystem.

SIXTH.- Diet, an active modulating factor of the intestinal microbiota, could also modulate equol production. Faecal cultures carried out in a dynamic intestinal model of the human proximal colon (TIM-2) showed that, compared to a balanced diet, equol production was doubled in a carbohydrate-rich diet, while it was reduced by half by a high-content protein diet. Those variations in equol production, however, lead to no significant changes in the numbers of equol-producing microorganisms in the cultures.

SEVENTH.- RT-PCR and RT-qPCR transcriptional analysis of all ORFs within the equol gene cluster of A. equolifaciens DSM19450T allowed to identify a unique operon-like structure constituted by 13 contiguous genes, among which tdr, ddr, and dzr were included. The presence of daidzein in cultures enhanced the expression of all genes and showed four differential expression patterns; these correlated with the position of putative promoter and terminator sequences within the operon.

EIGHTH.- Based on the A. equolifaciens DSM19450T genome sequence, four genes involved in equol production (racemase, tdr, ddr and dzr) were designed, synthesized, and cloned into model strains of Escherichia coli, Lactobacillus casei y L. lactis under the control of a promoter from the latter species. E. coli recombinant clones were able to produce equol from daidzein, while L. casei clones produced a small amount of equol only from the dihydrodaidzein intermediate; L. lactis clones did not produce equol. Biotechnological production in easily-cultivable heterologous hosts will enable a low-cost and safe synthesis of equol. However, equol biosynthesis in acid lactic bacteria (LAB) will require further studies, by employing, for instance, LAB-specific transcription and translation signals.

165

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Zheng, W., Ma, Y., Zhao, A., He, T., Lyu, N., Pan, Z., Mao, G., Liu,Y., Li, J., Wang, P., Wang, J., Zhu, B. y Zhang, Y. (2019). Compositional and functional differences in human gut microbiome with respect to equol production and its association with blood lipid level: a cross-sectional study. Gut Pathogens, 11(1), 20. doi:10.1186/s13099-019-0297-6.

Ziegler, R. G., Hoover, R. N., Pike, M. C., Hildesheim, A., Nomura, A. M. Y., West, D. W., Wu-Williams, A. H., Kolonel, L. N., Horn-Ross, P. L., Rosenthal, J. F. y Hyer, M. B. (1993). Migration patterns and breast cancer risk in Asian-American women. JNCI: Journal of the National Cancer Institute, 85(22), 1819- 1827. doi:10.1093/jnci/85.22.1819.

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ANEXOS ANNEXES Anexos ANEXO I.- Revisión bibliográfica nutrients

Review Equol: A Bacterial Metabolite from The Daidzein Isoflavone and Its Presumed Beneficial Health Effects

Baltasar Mayo 1,2,* , Lucía Vázquez 1,2 and Ana Belén Flórez 1,2 1 Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain; [email protected] (L.V.); abfl[email protected] (A.B.F.) 2 Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain * Correspondence: [email protected]; Tel.: +34-985893345

 Received: 22 August 2019; Accepted: 11 September 2019; Published: 16 September 2019 

Abstract: Epidemiological data suggest that regular intake of isoflavones from soy reduces the incidence of estrogen-dependent and aging-associated disorders, such as menopause symptoms in women, osteoporosis, cardiovascular diseases and cancer. Equol, produced from daidzein, is the isoflavone-derived metabolite with the greatest estrogenic and antioxidant activity. Consequently, equol has been endorsed as having many beneficial effects on human health. The conversion of daidzein into equol takes place in the intestine via the action of reductase enzymes belonging to incompletely characterized members of the gut microbiota. While all animal species analyzed so far produce equol, only between one third and one half of human subjects (depending on the community) are able to do so, ostensibly those that harbor equol-producing microbes. Conceivably, these subjects might be the only ones who can fully benefit from soy or isoflavone consumption. This review summarizes current knowledge on the microorganisms involved in, the genetic background to, and the biochemical pathways of, equol biosynthesis. It also outlines the results of recent clinical trials and meta-analyses on the effects of equol on different areas of human health and discusses briefly its presumptive mode of action.

Keywords: equol; daidzein; isoflavones; soy; soy products; gut metabolite; bioactive compound

1. Introduction Abundant epidemiological evidence suggests that diets rich in phytoestrogen-containing foods, such as soy and soy products, reduce the risk of a number of syndromes and chronic diseases, notably menopause symptoms in women, cardiovascular and neurodegenerative diseases, and certain types of cancer [1–3]. Isoflavones are non-nutritive phenolic compounds found in the roots and seeds of many plants, of these, soybeans are the richest source [4]. Soy isoflavones are phytoestrogens resembling 17-β-estradiol; although less active than the hormone, they show estrogen-like activity [5]. In plants, isoflavones are found mostly (>80%) in the form of glycoconjugates, i.e., the glucosides genistin, daidzin, and glycitin, and the corresponding acetyl and malonyl derivatives [6]. Glycosides are not readily absorbed in the gut and have only low-level estrogenic activity [7]. For isoflavones to become bioavailable and functional, these glycosides must be hydrolyzed into their corresponding isoflavone-aglycones, i.e., genistein, daidzein, and glycitein [8]. The amount of aglycones in plasma cannot be predicted from soy or isoflavone ingestion, as many intrinsic (genetic background, gut microbiota, bowel disease, age, sex, etc.) and extrinsic (isoflavone source, method of extraction, formulation, etc.) factors influence their bioavailability [9]. The plasma isoflavone concentration in humans is only 0.5–1.3% of that actually absorbed, much lower than in

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animalNutrients models 2019, (0.5–3.1%11, x FOR PEER for REVIEW rats and 3.1–26.0% in mice) [10]. Results obtained in animal2 of studies20 may not, therefore, be easily extrapolated to humans. Absorbed aglycones are metabolized mainly to glucuronidatedlower than in animal and sulphated models (0.5–3.1% derivatives for rats by and endogenous 3.1–26.0% phasein mice) I and[10]. phaseResults II obtained enzymes in [ 9,11]. Theyanimal may then studies be further may not, catabolized therefore, in be the easily liver orextrapolated secreted into to thehumans. bile, thusAbsorbed returning aglycones to the intestineare via themetabolized enterohepatic mainly circulation to glucuronidated [12]. and sulphated derivatives by endogenous phase I and phase II enzymes [9,11]. They may then be further catabolized in the liver or secreted into the bile, thus Certain aglycone conjugates may have estrogenic activity too, and serve as an intracellular reservoir returning to the intestine via the enterohepatic circulation [12]. for the release of free aglycones in target cells [13]. However, as for absorption, extrapolation of plasma Certain aglycone conjugates may have estrogenic activity too, and serve as an intracellular concentrationsreservoir for to thethe tissuerelease level of mayfree aglycones not be valid. in Indeed,target cells the identification[13]. However, and as quantificationfor absorption,of the isoflavoneextrapolation derivatives of plasma in target concentrations tissues has to rarely the tissu beene level determined may not [be14 valid.]. In humans, Indeed, the equol identification concentrations haveand been quantification reported to of range the isoflavone between derivatives 22–36 and in 456–559 target tissues nmol has/kg rarely in breast been adiposedetermined and [14]. glandular In tissuehumans, respectively equol [concentrations15,16]. have been reported to range between 22–36 and 456–559 nmol/kg in Unabsorbedbreast adipose isoflavones and glandular and tissue those respectively excreted by[15,16]. the biliary system to the intestine reach the colon where theyUnabsorbed are deconjugated isoflavones by bacterialand those enzymesexcreted by and the then biliary (re)absorbed system to the or intestine further metabolizedreach the colon [12 ,17]. In thewhere gut, they isoflavone are deconjugated aglycones by can bacterial be metabolized enzymes and by then intestinal (re)absorbed microbes or further via severalmetabolized reactions, [12,17]. In the gut, isoflavone aglycones can be metabolized by intestinal microbes via several including reduction, methylation/demethylation, hydroxylation/dihydroxylation, and C-ring cleavage reactions, including reduction, methylation/demethylation, hydroxylation/dihydroxylation, and (FigureC-ring1). cleavage (Figure 1).

FigureFigure 1. Metabolism 1. Metabolism of of the the isoflavone isoflavone glucosideglucoside daidzein daidzein by by the the human human gut gutmicrobiota microbiota and equol and equol biosynthesisbiosynthesis pathway. pathway

2. Equol2. Equol

ExtractedExtracted from from the the urine urine of of pregnant pregnant mares mares backback in 1932, 1932, equol equol [C [C15H1512HO(OH)12O(OH)2] was2] wasthe first the first isoflavonoidsisoflavonoids to be to identified be identified [18]. Later,[18]. Later, in 1982 in it1982 was it the was first the isoflavone-derived first isoflavone-derived compound compound detected in humandetected urine andin bloodhuman (reviewed urine and by Setchellblood (reviewed and Clerici by [ 19Setchell]). Equol and is anClerici isoflavone-derived [19]). Equol is metabolite an formedisoflavone-derived from daidzin/daidzein metabolite by bacteriaformed from in the daidzin/da distal regionidzein of theby bacteria small intestine in the distal and colonregion [ 19of ].the From a chemicalsmall viewpoint, intestine and equol colon (4’,7-isoflavandiol) [19]. From a chemical is viewpo an isoflavaneint, equol phenolic (4',7-isoflavandiol) compound is withan isoflavane a non-planar phenolic compound with a non-planar structure, which might be responsible for its physiological structure, which might be responsible for its physiological activities [20]. Equol is optically active with activities [20]. Equol is optically active with an asymmetric carbon atom at the C3 position giving an asymmetricrise to R(-)- carbon and S(-)-equol atom at enantiomers. the C3 position However, giving only rise toS(-)-equolR(-)- and hasS(-)-equol been detected enantiomers. as a result However, of only S(-)-equol has been detected as a result of bacterial daidzein conversion [21–23]. Equol is more stable, more easily absorbed, and has a lower clearance than its precursor molecule daidzein [24]. It also shows stronger estrogenic activity than any other isoflavone or isoflavone-derived metabolite [23,25].

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As for other isoflavones, equol also shows anti-androgenic activity by binding to and sequestering 5α-dihydrotestosterone [26]. In addition, it is the isoflavone-derived compound with the strongest antioxidant activity [27,28]; antioxidants are thought to have a prominent role in the onset and progress of different chronic diseases, including cancer [29].

2.1. Equol Production Phenotype Equol is produced from the isoflavone daidzein in the gut of humans and animals by certain bacterial biotypes; those involved might differ between individuals [30]. All the animal species tested (mouse, rat, sheep, cow, goat, chicken, and fowl) have been shown to produce equol in response to soy or daidzein consumption [21]. However, it is produced by only 25–50% of human subjects (so-called equol producers); the percentage depends on the community in question and the dietary habits of its members [31–34]. In contrast, a majority of humans (80–90%) that do not produce equol (equol non-producers), convert a large part of daidzein into O-desmethylangolensin (O-DMA), a metabolite with no estrogenic activity [35,36]. O-DMA and equol are likely produced by different bacterial taxa. Both observational and interventional studies have returned inconsistent results on the stability of the equol production phenotype in humans. Some authors propose equol production status to be rather stable [24,37], suggesting it to be under some degree of genetic control. However, other studies have reported the conversion, at small rates, of producers to non-producers and vice versa [38,39]. Thus, equol production appears to be stable in some but not all individuals. The frequency of equol producers among vegetarians has been reported significantly higher than among non-vegetarians (59% versus 25%) [34], suggesting that dietary components from plants other than soy itself may promote the ability to produce equol. Equol is not detected in the urine and plasma of most infants under one year of age [40,41], indicating that equol-producing bacteria are latecomers to the gastrointestinal ecosystem. The ability to produce equol may be influenced by shared environmental factors, although weak positive correlations between mothers and children have been reported [42,43]. Another topic of debate is whether dietary constituents other than isoflavones enhance equol formation in equol producers [38,44–46]. The consumption of resistant starch (together with daidzein) by ovariectomized mice has been correlated with enhanced equol excretion [47]. Similarly, the consumption of daidzein and lactulose has been reported to promote equol production in sows [48], and carbohydrate-rich diets have been shown to stimulate equol production in human fecal cultures [49]. The combined consumption of milk and dairy products with daidzein is also significantly correlated with equol excretion concentrations [50]. The antibiotic treatment of fecal cultures from different subjects has been shown to both increase and reduce equol production [46], again suggesting differences between people in terms of the equol-producing microorganisms carried. Together, these findings suggest that the equol production phenotype and equol production itself are modified by dietary habits.

2.2. Equol-Producing Microorganisms It is well established that equol is formed from daidzein by gut bacteria [30,51]. However, our knowledge of the actual microorganisms involved is still limited [52]. Bacterial mixtures of different taxa capable of producing equol from daidzein have been described [49,53]. A few strains are known to convert daidzein into dihydrodaidzein although they cannot produce equol, while others can convert dihydrodaidzein into equol but do not act on daidzein (Table 1). In these cases, equol production has been detected by combining dihydrodaidzein producers (e.g., Lactobacillus sp. Niu-O16) with dihydrodaidzein utilizers (e.g., Eggerthella sp. Julong 732) [53]. In recent decades, a number of strains (from humans and animals) capable of forming equol have finally been identified (Table 1). Even, a few equol-producing bacteria can also metabolize genistein to generate 5-hydroxy equol [54–56], a compound highly similar to equol from a chemical point of view and with greater antioxidant activity than genistein [57]. Most of the equol-producing microbes isolated so far belong to the family Coriobacteriaceae [52]. Members of this family are also involved in the catabolism of cholesterol-derived compounds such as

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Nutrients 2019, 11, 2231 bile acids and corticoid hormones, hinting at their functional specialization in the gut [58]. The family Coriobacteriaceae includes genera such as Adlercreutzia, Assacharobacter, Eggerthella, Enterorhabdus, Paraeggerthela, and Slackia [52]. Among these, species such as Adlercreutzia equolifaciens, Asaccharobacter celatus, Enterorhabdus mucosicola, Slackia isoflavoniconvertens, and Slackia equolifaciens are reported to be equol producers (Table 1). Some strains have been identified only at the genus level; e.g., Eggerthella sp. YY7918, Paraeggerthella sp. SNR40-432, and Slackia sp. NATTS (Table 1). In spite of this, it is not yet sure whether equol production in the Coriobacteriaceae is a family-, species-, or strain-specific trait [59]. A few equol-producing strains of other taxa from either intestinal or food origin have recently been identified (Table 1), including Bifidobacterium breve ATCC 15700T, Bifidobacterium longum BB536, Lactobacillus intestinalis JCM 7548, Lactobacilllus paracasei CS2, Lactobacillus sakei CS3, Lactococcus garvieae 20-90, Pediococcus pentosaceus CS1, and Proteus mirabilis LH-52.

Table 1. Bacterial species and strains involved in the metabolism of equol or its intermediate precursors from daidzein.

Species Strain/s Origin Reference Adlercreutzia equolifaciens FJC-B9 T Human feces Maruo et al. [60] Asaccharobacter celatus do03 T Rat cecum Minamida et al. [61] Bifidobacterium breve ATCC15700 T Human intestine Elghali et al. [62] Bifidobacterium longum BB536 Human feces Elghali et al. [62] Catenibacterium sp. D1 Human feces Yu et al. [63] Clostridium sp. HGH6 a Human feces Hur et al. [64] Clostridium-like sp. TM-40 a Human feces Tamura et al. [65] Eggerthella sp. YY7918 Human feces Yokoyama and Suzuki [66] Eggerthella sp. D2 Human feces Yu et al. [63] Eggerthella sp. Julong 732 b Human feces Wang et al. [67] SNR48-44, SNR44-10, SNR45-571, Eggerthella-like bacteria Stinky tofu Abiru et al. [54] SNR46-41, SNR48-350 Enterorhabdus mucosicola Mt1B8 T Mouse ileal mucosa Matthies et al. [68] Lactobacillus sp. Niu-O16 a Bovine rumen Wang et al. [67] Lactobacillus paracasei CS2 (JS1) Human feces Kwon et al. [69] Lactobacillus sakei/graminis CS3 Human feces Kwon et al. [69] Lactobacillus intestinalis JCM 7548 Rat feces Heng et al. [70] Lactococcus garvieae 20-92 Human feces Uchiyama et al. [71] Paraeggerthella sp. SNR40-432 Stinky tofu Abiru et al. [54] Pediococcus pentosaceus CS1 Human feces Kwon et al. [69] Proteus mirabilis LH-52 Rat intestine Guo et al. [72] Slackia equolifaciens DZE Tc Human feces Jin et al. [73] Slackia isoflavoniconvertens HE8 Tc Human feces Matthies et al. [55] Slackia sp. NATTS Human feces Tsuji et al. [74] a Daidzein to dihydrodaidzein only. b Equol from dihydrodaidzein only. c These strains are also able to produce 5-hydroxy equol from the isoflavone genistein. The T superscript denotes the isolate as the species type strain.

2.3. Molecular Aspects of Equol Formation The bacterial biosynthesis of equol from daidzein proceeds via a series of consecutive reduction reactions (catalyzed by three reductases), involving the production of the intermediate compounds dihydrodaidzein and tetrahydrodaidzein (Figure 1). In L. garvieae, the genes involved in equol production are found in a 10 kbp operon-like structure [75–77]. At least three genes encoding a daidzein-dependent NADP reductase (dzr), a dihydrodaidzein reductase (ddr), and a tetrahydrodaidzein reductase (tdr), have been reported to be required for equol production in this bacterium. A fourth enzyme with dihydrodaidzein racemase activity, encoded immediately upstream of the reductase genes in the equol cluster, has been shown to be necessary for efficient equol production by L. garvieae [77]. All reactions in the pathway seem to be reversible [75]. More recently, next generation sequencing techniques have helped characterize the genomes of other equol-producing strains [78,79], which helped to revealed the genetic basis of the biochemical pathways involved in the synthesis of equol. In S. isoflavoniconvertens, the equol cluster is about 10.5 kbp in length and contains eight genes [80]. As in L. garvieae, the reductase enzymes in S. isoflavoniconvertens are encoded by homologous dzr, ddr, and tdr genes [80]. Equivalent genes encoding reductases similar to those of L. garvieae and S. isoflavoniconvertens

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Nutrients 2019, 11, 2231 have also been identified in Slackia sp. NATTS [81], A. equolifaciens [78], and Eggerthella sp. YY7918 [82]. Interestingly, the L. garvieae genes encoding the equol-related reductases have been reported similar to those in the Coriobacteriaceae (such as S. isoflavoniconvertens, Eggerthella sp., and Slackia sp. NATTS), arguing strongly for the recent horizontal transfer of the equol genetic makeup from a member of this family. This is further supported by the specific codon usage and high GC content (68%) of the equol-associated genes in L. garvieae [75,76], which greatly exceeds the genomic GC content of this species (39%). The genetic framework and the biochemical pathways of equol production in other non-Coriobacteriaceae species have yet to be determined. The proteomic analysis of S. isoflavoniconvertens grown with daidzein has shown overexpression of the reductases and five other proteins encoded by genes located within the equol gene cluster [80]. Congruently, all enzymes of the cluster might be somehow involved in equol production and can be regulated in a coordinated manner. Indeed, the expression of 13 contiguous genes in the equol cluster of A. equolifaciens has recently been shown enhanced during the growth of this bacterium in the presence of daidzein, during which dzr, ddr, and tdr were the most strongly expressed genes [83]. Four expression patterns of transcription for the genes of the A. equolifaciens equol cluster were identified, although the operon seemed to be transcribed as a single RNA transcript [83]. The roles of other genes in the operon, (of which at least three encode flavoproteins that might be involved in oxidation-reduction reactions), and their regulatory mechanisms, are yet to be determined. In S. isoflavoniconvertens, the first enzyme of the pathway, daidzein reductase, has also been shown to participate in the transformation of genistein into dihydrogenistein, a key step in the formation of 5-hydroxy equol [80], a compound also produced by E. mucosicola [68]. The biotechnological synthesis of equol by anaerobic coriobacteria requires long culture times and is expensive. Cloning and expressing the genetic machinery of equol production in heterologous hosts might circumvent these challenges [80,81] while helping to reveal the function of all the determinants in the cluster. The genes encoding the three reductases of L. garvieae (dzr, ddr, and tdr) were soon cloned and expressed in Escherichia coli [75,76]. The same genes from S. isoflavoniconvertens and Eggerthella sp. YY7918 have been also cloned individually and expressed in E. coli [80,82]. This strategy identified the involvement of the S. isoflavoniconvertens reductases in the conversion of genistein into 5’-hydroxy-equol [80,84]. Expression of the genes in E. coli further allowed the development of recombinant strains with improved S(-)-equol production capacity [85,86]. A low production yield by fermentation has been reported when using recombinant microorganisms, perhaps due to the low solubility of isoflavones in aqueous systems. This problem has been recently overcome by adding hydrophilic polymers to the cultures [87]. Large scale production would surpass the current equol shortage, supplying enough for interventional studies that could assess its efficacy.

2.4. Equol-Producing Populations in the Human Gut Since the gut microbiota plays an important role in the metabolism of soy isoflavones, understanding the role of soy and its components in influencing and modulating the gut microbiota is vital if we are to learn the mechanisms of action of soy’s bioactive compounds and promote their rational use in functional foods [88,89]. Equally important will be to know the types and numbers of microorganisms involved in the synthesis of equol present in the microbiota of different individuals. To that aim, based on conserved sequences of genes involved in equol synthesis from known equol-producing species, oligonucleotide primers have been developed to detect [90] and quantify [91] equol-producing microbes in samples of fecal origin. These primers have already been used to amplify reductase-encoding genes related to those of A. equolifaciens and S. isoflavoniconvertens in feces and fecal cultures [90,91]. However, no amplicons were obtained when DNA from fecal samples of certain equol producers was used as a template, suggesting the involvement of other yet unknown unrelated taxa in equol formation in these individuals [90,91]. Similar copy numbers of both tdr and ddr genes, about 4–5 log10 copies per gram of feces, have been reported [91]. As these are single-copy genes, an equal number of equol-producing bacteria must be expected. Indeed, these amounts are

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Nutrients 2019, 11, 2231 within the usual numbers for Coriobacteriaceae species in feces as quantified by 16S rRNA gene amplification [92–94]. Surprisingly, no significant increases in the copy number of equol-related genes (and thus bacteria) has ever been observed during isoflavone interventions [91] or after in vitro culturing of fecal samples, even under conditions in which equol production is favored (such as in carbohydrate-rich diets) (Vázquez et al., unpublished). These results imply that equol-producing bacteria are not positively selected for by isoflavones. It is also surprising that the fact that genes involved in equol production and equol-producing bacterial numbers have been reported in both equol-producing and non-producing individuals [44,91]. Altogether these results suggest that more studies will be required if we are to understand the composition and changes in equol-producing populations in the gut. As minority populations, deciphering the interaction of the equol producing microbes with majority and pivotal microbial communities within the gut ecosystem is paramount.

3. Soy, Soy Isoflavones, Equol, and Health In East Asian countries, climacteric vasomotor symptoms during menopause in women are less severe than in Western women and the incidence of cardiovascular disease, osteoporosis, mental disorders, and certain types of cancer is about two- to four-fold of that seen in the West[1–3,95–97]. Alongside genetic factors, this large difference is assumed to have a nutritional basis. Isoflavones are an important component of Asian diets (15–50 mg day versus <2 mg in Western countries) [98–101], and observational and epidemiological studies have correlated a high intake of soy and isoflavones with reduced menopause symptoms, increased bone formation, reduced bone resorption, improved learning, and a reduced risk of prostate, colon, and breast cancer [102–105]. In vitro laboratory studies and animal interventions can predict the impact of isoflavones on human health, but only human trials can provide proof. However, most current human interventions involving isoflavones have suffered from small sample sizes, short trial durations, lack of appropriate controls, the use of isoflavones from various sources, supplements with different aglycone contents, and other methodological flaws [106]. Not surprisingly, this has led to inconsistent results being reported [96,107–111]. Indeed, most reviews and meta-analyses report the results of soy and isoflavone intervention studies to be far from conclusive [1,112–117]. As a result, regulatory agencies usually conclude there to be no scientifically sound evidence of isoflavones reducing the risks and symptoms of any disease [14,106,118]. In addition to the effect of genetic variation on the phenotypic expression of human disease [119,120], interpersonal differences in the intestinal microbiota may also account (at least in part) for the discrepancies seen [121,122]. Such differences could give rise to different microbial isoflavone-derived metabolites being produced [12,33], which might explain the lack of effectiveness in some studies. In particular, there has been much speculation regarding the reason why just a fraction (25 to 50%) of the human population produces equol. Conceivably, these subjects might be the only ones who would benefit from soy or isoflavone consumption [111,123,124]. To test this hypothesis, the categorization of the individuals in isoflavone trials by their equol-producing phenotype is pivotal. This only began in recent years [32,96,124–126] and no firm conclusions have yet been drawn. Indeed, the results of many studies have been very conflicting [108–111,127–130]. In contrast to their possible health benefits, the anti-estrogenic properties of isoflavones might also cause them to act as unwanted endocrine disruptors [131]. In vitro and animal studies both report isoflavones able to interfere with different checkpoints of the hypothalamic/pituitary/thyroid system [132]. This could have a huge repercussion on thyroid homeostasis. Further, the estrogenic activity of isoflavones (and thus equol) could pose a potential hazard by promoting certain types of tumor [133]. However, the scientific evidence supporting their having any harmful consequences is also inconclusive [14,106].

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3.1. Equol, Menopause, and the Cardiovascular System Soy intake has been correlated with fewer hot flushes and night sweats during menopause [1,134]. In addition, there is growing evidence that soy and soy isoflavones may help regulate vasoactivity [135], as well as lipid metabolism and cholesterol transport [136–138]. Equol offers an alternative for the management of menopausal symptoms, extending the otherwise reduced benefits of soy isoflavones or isoflavone supplement consumption to beyond equol producing women. Intervention trials in humans have frequently focused on non-equol-producing populations [107,139–141]. Compared to placebo controls, beneficial effects have been reported for women taking an S(-)-equol supplement (10 to 30 mg daily for 8–12 weeks) with respect to the major menopausal symptoms (hot flushes) [139,142] and arterial stiffness [143]. A recent meta-analysis also revealed a significant reduction in hot flash scores (incidence and/or severity) following equol supplementation in both equol-producing and non-producing menopausal women [144]. Thus, equol might serve as a new, promising, and safe therapeutic option to be used as complementary therapy for women with vasomotor symptoms. In the Orient, age-adjusted mortality rates of cardiovascular diseases are lower of that seen in Western countries and inversely correlated to isoflavone excretion in urine [98,134]. Recent observational studies and short-term randomized controlled trials have correlated equol with a reduced risk of coronary heart disease via its enhanced anti-atherogenic potential and the improvement of arterial stiffness [145]. Interventions with natural equol in overweight Japanese men and women suggested it may help in the prevention of cardiovascular diseases by lowering low-density lipoprotein cholesterol (LDL-C) levels and improving the cardio-ankle vascular index (a blood pressure-independent index of arterial stiffness) [146]. However, no vascular benefits (arterial stiffness, blood pressure, endothelial function, and nitric oxide formation) were observed in equol non-producing men after the acute intake of equol (40 mg) [107].

3.2. Equol and Bone Health Isoflavones have been repeatedly reported to help prevent osteoporosis, a major problem for menopausal women [104,130,147,148]. The exact mechanism by which isoflavones preserve bone health is not completely understood. It seems that isoflavones trigger the activity and proliferation of osteoblasts via insulin-like growth factor 1 (IGF-1), a key factor in maintaining bone mass against the action of osteoclasts [149]. In a meta-analysis study, Wei and coworkers have found that, as compared to baseline levels, intake of soy isoflavone supplements significantly increased bone mineral density and decreased the bone resorption marker urinary deoxypyridinoline [104]. However, in the same study, no significant effect on serum bone alkaline phosphatase activity (which is involved in bone formation) was observed. In mice, equol has been shown to reduce the expression of genes associated with the inhibition of bone formation, osteoclast and immature osteoblast specificity, and cartilage destruction [150]. In humans, the treatment of postmenopausal women with 10 mg/day of equol for one year prevented a reduction in bone mineral density in the entire body [143]. This work further showed that equol supplementation markedly inhibited bone resorption, as demonstrated by reduced urinary deoxypyridinoline excretion concentrations.

3.3. Equol and Cancer The incidence of prostate, colon, and breast cancers is much lower in East Asian countries than in the West [2,3,93,96,151,152]. Although environmental factors are thought to contribute strongly to the development of tumors, Asian immigrants to Western countries who change their dietary habits suffer from these forms of cancer at similar rates to Westerners, which suggests that isoflavones, via soy consumption, might be related to this reduction in risk [96]. Further, recent evidence suggests there is a reduced risk of developing breast cancer if soy was consumed during childhood and/or adolescence [153].

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Only a few observational studies have investigated the influence of equol and the equol-producer status on breast cancer incidence or markers of breast cancer risk. Certainly, the plasma concentration of equol in women with breast cancer has been found similar to that of healthy controls [154], and no association between equol-producer status and breast cancer risk has ever been established [155]. Indeed, contradictory results have also been obtained, even from the same human group (the EPIC-Norfolk Cohort) [10,156]. Controversial results have also been reported regarding breast density in women (another marker of breast cancer) and soy food or soy supplement consumption, and the equol producer or non-producer phenotype [155,157]. In men, the ability to produce equol or equol itself has been suggested to help in reducing the incidence of prostate cancer [39]. The results also suggest that a diet based on soybean isoflavones could be useful in preventing prostate cancer.

3.4. Equol and the Central Nervous System Epidemiological studies reveal lower rates of dementia in East Asian populations [158]. Studies on the effects of isoflavones on the gut-brain axis in humans have focused mostly on cognitive functions. In general, beneficial effects have been reported [159–163]. The long-term administration of soy or extracted isoflavones has been associated with improved learning, logical thinking, and planning ability in menopausal women. However, inconclusive findings on the neuroprotective effects of isoflavones and other phytoestrogens have also been reported [164]. Evidence is, however, accumulating that atherosclerosis and arterial stiffness are positively associated with cognitive decline. Sekikawa et al. [145], who showed that S-equol was anti-atherogenic and could improve arterial stiffness, reported in their review that equol may help prevent cognitive impairment/dementia.

3.5. Equol and Other Health Benefits A topical equol intervention suggested equol to have an anti-aging effect on the skin of postmenopausal women, reducing wrinkle area and depth [165]. More recently, equol application to the skin has also been associated with an improvement in skin roughness, texture, and smoothness, and in some epigenetic molecular markers (LINE-1 methylation and telomere length) in skin cells [166]. Not surprisingly, the compound has recently attracted substantial attention of the cosmetic industry. Equol has also been suggested to modulate obesity and diabetes type-2 by controlling the glycemic index [146,167], and to ameliorate chronic kidney disease [103].

4. Mechanistic Mode of Action of Equol The mechanistic mode of action of equol is not yet completely understood. Studies have mostly been done through in vitro assays using concentrations higher than those found under physiological conditions, thus limiting the provision of robust and definitive conclusions. Further, equol is found in plasma mainly as a 7-O-glucuronide derivative [168], which makes it difficult to discern the biologically-active form(s) at tissue and cellular levels. In spite of these deficits, evidence from experimental studies suggests that equol may act in multiple ways [169]. Based on its structural similarity to 17-β-estradiol, equol binds to both estrogen receptors (ERs) α (ERα) and β (ERβ—the preferred target) with greater affinity than its precursor daidzein, and to a degree comparable to that of genistein [170,171]. As ERs are not equally distributed among the different tissues, equol might have different effects depending on the ratio of ERα and ERβ isoforms present. Whether it acts as an agonist or an antagonist may further depend on the level of endogenous estrogens present, as they bind to both receptors more tightly [172]. The antioxidant activity of equol seems to be mostly mediated by its interaction with the ERβ [27], which induces the extracellular signal-regulated protein kinases (ERK1/2) and the NF-κB peptide, factors that control transcription, cytokine production, and cell survival [173]. Isoflavones and equol may not act as antioxidants themselves but rather by triggering cell signaling pathways leading to changes in the expression of cellular enzymes such as superoxide dismutase, catalase, and glutathione peroxidase (all involved in counteracting oxidative stress) [29].

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Mechanistically, equol’s influence on transcription seems to proceed through the activation of the transactivation function AF-1 [174]. Another mode of action of equol underlying several physiological effects may relate to epigenetic mechanisms, including DNA methylation, histone modification, and microRNA regulation [175]. Equol has been reported to induce acute endothelium- and nitric oxide (NO)-dependent relaxation of the aortic rings, and is a potent activator of the human and mouse pregnane X receptor (PXR), a steroid and xenobiotic sensing protein in the nucleus [176]. Further, it has been proposed that it modulates endothelial redox signaling and NO release, involving the transactivation of the epidermal growth factor receptor kinase (EGFRK) and the reorganization of the F-actin cytoskeleton [177]. Equol has also been shown to prevent (at physiological concentrations) oxidized LDL-stimulated apoptosis in human umbilical vein endothelial cells [178], and to reduce the oxidative stress induced by lipopolysaccharides in chicken macrophages [179]. These activities may provide the basis for therapeutic strategies, for instance by restoring endothelial function in cardiovascular diseases. An improvement in atherosclerosis has also been reported via equol attenuating ER stress, mediated by the activation of the NF-E2 p45-related factor 2 (Nrf2) signaling pathway [180]. The cancer-protective effects of isoflavones and equol have been attributed to a variety of signaling pathways, including the regulation of the cell cycle (by reducing the activity of the cyclin B/CDK complex) [181], the inhibition of cell proliferation (by, for instance, reducing activity of topoisomerase II) [182], the induction of apoptosis [173,183,184], and the degradation of androgen receptor by S-phase kinase-associated protein 2 (PKAP2) [185]. The anti-prostate cancer activity of equol in cell cultures has been proposed associated with activation of FOXO3a (one of the forkhead-family factors of transcription involved in apoptosis) via protein kinase B (Akt)-specific signaling transduction pathway, and with the inhibition of the expression of the MDM2 complex (a negative regulator of tumor suppressor p53) [186,187], plus the inhibition of the degradation of the androgen receptor [185]. Diabetes and other metabolic disorders may be influenced by equol via its preventing glucagon-like peptide 1 (GLP-1) secretion from the GLUTag cells [188]. The modulation of glucose-induced insulin secretion and the suppression of glucagon release (from the α- and β-pancreatic cells, respectively) by GLP-1 in response to the ingestion of nutrients have been firmly established [189]. Equol can also prevent hypoglycemia by activating cAMP signaling at the plasma membrane of INS-1 pancreatic β-cells [190]. Finally, it has been reported to significantly increase the expression of genes coding for collagen, elastin, and tissue inhibitor of metalloproteases, while reducing the expression of metalloproteinases [191]. All these factors contribute towards an improvement of the skin’s antioxidant status, delaying aging. However, despite of all the knowledge gathered by these in vitro observations, the effects of equol on human health in vivo, and their magnitude, are yet to be confirmed.

5. Conclusions In summary, current interest in dietary isoflavones has been driven by epidemiological studies, suggesting that diets rich in phytoestrogens are beneficial to human health. Soy isoflavones and isoflavone-derived metabolites are structurally similar to estrogen and might have some of its effects. Equol is a key isoflavone-derived metabolite with estrogenic and antioxidant activities. Studies examining the influence of equol and the equol-producer status on several disease conditions have been inconclusive, and there is an urgent need for large-scale, well designed, randomized, double blind, placebo-controlled human trials. Further knowledge is also required on the changes in metabolic markers induced by isoflavones/equol interventions. Understanding the gut microbial populations involved in equol biosynthesis, and their regulatory mechanisms, is also pivotal for maximizing endogenous equol production. Identification of the actual compound(s) with a role in the signaling cascades underlying the involved cellular and physiological processes would contribute greatly to the functional characterization of the role of this bioactive metabolite. The exploitation of equol-producing microorganisms or their genetic machinery for the biotechnological production of this bioactive agent would allow the use of equol in large-scale interventional trials. This would

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Nutrients 2019, 11, 2231 ultimately serve to test the real involvement of equol in human health. Finally, well characterized equol-producing strains could be used in the future as probiotics for animals and humans aimed as a means of increasing equol production in the gut.

Funding: This research was partly funded by grants from the Spanish Ministry of Economy and Competitiveness (MINECO) (AGL2011-24300-ALI and AGL-2014-57820-R) and the Principality of Asturias (GRUPIN14-137 and IDI/2018/000114). L.V. was funded by a contract from MINECO within the FPI Program (BES-2015-072285). Conflicts of Interest: The authors declare no conflict of interest.

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168. Gardana, C.; Simonetti, P. Long-term kinetics of daidzein and its main metabolites in human equol-producers after soymilk intake: Identification of equol-conjugates by UPLC-orbitrap-MS and influence of the number of transforming bacteria on plasma kinetics. Int. J. Food Sci. Nutr. 2017, 68, 496–506. [CrossRef][PubMed] 169. Chadha, R.; Bhalla, Y.; Jain, A.; Chadha, K.; Karan, M. Dietary soy isoflavone: A mechanistic insight. Nat. Prod. Commun. 2017, 12, 627–634. [CrossRef][PubMed] 170. Paterni, I.; Granchi, C.; Katzenellenbogen, J.A.; Minutolo, F. Estrogen receptors alpha (ERα) and beta (ERβ): Subtype-selective ligands and clinical potential. Steroids 2014, 90, 13–29. [CrossRef] 171. Lehmann, L.; Esch, H.L.; Wagner, J.; Rohnstock, L.; Metzler, M. Estrogenic and genotoxic potential of equol and two hydroxylated metabolites of daidzein in cultured human Ishikawa cells. Toxicol. Lett. 2005, 158, 72–86. [CrossRef] 172. Hertrampf, T.; Schmidt, S.; Laudenbach-Leschowsky, U.; Seibel, J.; Diel, P. Tissue-specific modulation of cyclooxygenase-2 (Cox-2) expression in the uterus and the v. cava by estrogens and phytoestrogens. Mol. Cell. Endocrinol. 2005, 243, 51–57. [CrossRef] 173. Yang, Z.; Zhao, Y.; Yao, Y.; Li, J.; Wang, W.; Wu, X. Equol induces mitochondria-dependent apoptosis in human gastric cancer cells via the sustained activation of ERK1/2 pathway. Mol. Cells 2016, 39, 742–749. [CrossRef] 174. Shinkaruk, S.; Durand, M.; Lamothe, V.; Carpaye, A.; Martinet, A.; Chantre, P.; Vergne, S.; Nogues, X.; Moore, N.; Bennetau-Pelissero, C. Bioavailability of glycitein relatively to other soy isoflavones in healthy young Caucasian men. Food Chem. 2013, 135, 1104–1111. [CrossRef][PubMed] 175. Rietjens, I.M.; Sotoca, A.M.; Vervoort, J.; Louisse, J. Mechanisms underlying the dualistic mode of action of major soy isoflavones in relation to cell proliferation and cancer risks. Mol. Nutr. Food Res. 2013, 57, 100–113. [CrossRef][PubMed] 176. Joy, S.; Siow, R.C.; Rowlands, D.J.; Becker, M.; Wyatt, A.W.; Aaronson, P.I.; Coen, C.W.; Kallo, I.; Jacob, R.; Mann, G.E. The isoflavone equol mediates rapid vascular relaxation: Ca2+-independent activation of endothelial nitric-oxide synthase/Hsp90 involving ERK1/2 and Akt phosphorylation in human endothelial cells. J. Biol. Chem. 2006, 281, 27335–27345. [CrossRef][PubMed] 177. Rowlands, D.J.; Chapple, S.; Siow, R.C.; Mann, G.E. Equol-stimulated mitochondrial reactive oxygen species activate endothelial nitric oxide synthase and redox signaling in endothelial cells: Roles for F-actin and GPR30. Hypertension 2011, 57, 833–840. [CrossRef][PubMed] 178. Kamiyama, M.; Kishimoto, Y.; Tani, M.; Utsunomiya, K.; Kondo, K. Effects of equol on oxidized low-density lipoprotein-induced apoptosis in endothelial cells. J. Atheroscler. Thromb. 2009, 16, 239–249. [CrossRef] [PubMed] 179. Gou, Z.; Jiang, S.; Zheng, C.; Tian, Z.; Lin, X. Equol inhibits LPS-induced oxidative stress and enhances the immune response in chicken HD11 macrophages. Cell. Physiol. Biochem. 2015, 36, 611–621. [CrossRef] [PubMed] 180. Zhang, T.; Hu, Q.; Shi, L.; Qin, L.; Zhang, Q.; Mi, M. Equol attenuates atherosclerosis in apolipoprotein E-deficient mice by inhibiting endoplasmic reticulum stress via activation of Nrf2 in endothelial cells. PLoS ONE 2016, 11, e0167020. [CrossRef][PubMed] 181. Casagrande, F.; Darbon, J.M. Effect of structurally related flavonoids on cell cycle progression of human melanoma cells: Regulation of cyclin-dependent kinases CDK11 and CDK2. Biochem. Pharmacol. 2001, 61, 1205–1215. [CrossRef] 182. Mizushina, Y.; Shiomi, K.; Kuriyana, I.; Takahashi, Y.; Yoshida, H. Inhibitory effect of a major soy isoflavone, genistein, on human DNA topoisomerase II activity and cancer cell proliferation. Int. J. Oncol. 2013, 43, 1117–1124. [CrossRef] 183. Ono, M.; Ejima, K.; Higuchi, T.; Takeshima, M.; Wakimoto, R.; Nakano, S. Equol enhances apoptosis-inducing activity of genistein by increasing Bax/Bcl-xL expression ratio in MCF-7 human breast cancer cells. Nutr. Cancer 2017, 69, 1300–1307. [CrossRef] 184. Kim, E.Y.; Shin, J.Y.; Park, Y.J.; Kim, A.K. Equol induces mitochondria-mediated apoptosis of human cervical cancer cells. Anticancer Res. 2014, 34, 4985–4992. [PubMed] 185. Itsumi, M.; Shiota, M.; Takeuchi, A.; Kashiwagi, E.; Inokuchi, J.; Tatsugami, K.; Kajioka, S.; Uchiumi, T.; Naito, S.; Eto, M.; et al. Equol inhibits prostate cancer growth through degradation of androgen receptor by S-phase kinase-associated protein 2. Cancer Sci. 2016, 107, 1022–1028. [CrossRef]

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186. Lu, Z.; Zhou, R.; Kong, Y.; Wang, J.; Xia, W.; Guo, J.; Liu, J.; Sun, H.; Liu, K.; Yang, J.; et al. S-equol, a secondary metabolite of natural anticancer isoflavone daidzein, inhibits prostate cancer growth in vitro and in vivo, though activating the Akt/FOXO3a pathway. Curr. Cancer Drug Targets 2016, 16, 455–465. [CrossRef] [PubMed] 187. Yang, Z.P.; Zhao, Y.; Huang, F.; Chen, J.; Yao, Y.H.; Li, J.; Wu, X.N. Equol inhibits proliferation of human gastric carcinoma cells via modulating Akt pathway. World J. Gastroenterol. 2015, 21, 10385–10399. [CrossRef] [PubMed] 188. Harada, K.; Sada, S.; Sakaguchi, H.; Takizawa, M.; Ishida, R.; Tsuboi, T. Bacterial metabolite S-equol modulates glucagon-like peptide-1 secretion from enteroendocrine L cell line GLUTag cells via actin polymerization. Biochem. Biophys. Res. Commun. 2018, 501, 1009–1015. [CrossRef][PubMed] 189. Andersen, A.; Lund, A.; Knop, F.K.; Vilsbøll, T. Glucagon-like peptide 1 in health and disease. Nat. Rev. Endocrinol. 2018, 14, 390–403. [CrossRef][PubMed] 190. Horiuchi, H.; Usami, A.; Shirai, R.L.; Harada, N.; Ikushiro, S.; Sakaki, T.; Nakano, Y.; Inui, H.; Yamaji, R. S-Equol activates cAMP signaling at the plasma membrane of INS-1 pancreatic β-cells and protects against streptozotocin-induced hyperglycemia by increasing β-cell function in male mice. J. Nutr. 2017, 147, 1631–1639. [CrossRef][PubMed] 191. Gopaul, R.; Knaggs, H.E.; Lephart, E.D. Biochemical investigation and gene analysis of equol: A plant and soy-derived isoflavonoid with antiaging and antioxidant properties with potential applications. Biofactors 2012, 38, 44–52. [CrossRef][PubMed]

© 2019 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 (http://creativecommons.org/licenses/by/4.0/).

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ANEXO II.- Informe sobre la calidad de los artículos

La información sobre la calidad de los artículos que componen esta memoria de Tesis Doctoral ha sido recogida de la “Web of Science” (www.recursoscientificos.fecyt.es). Se han recopilado los siguientes parámetros para cada artículo: el área SCI a la que se encuentra asociada la revista; el factor de impacto (FI) de la revista correspondiente al año de publicación del artículo o en caso de los artículos más recientes, a los últimos datos publicados por “Journal Citation Reports” (año 2019); el cuartil (Q) de la revista dentro de cada área, calculado en función de su factor de impacto; y las citas que representa el número de veces que ha sido citado cada artículo obtenidas de “Scopus” hasta el momento de la escritura de la Tesis (Septiembre del 2020).

. Artículo 1.- Vázquez, L., Guadamuro, L., Giganto, F., Mayo, B. y Flórez, A. B. (2017). Development and use of a real-time quantitative PCR method for detecting and quantifying equol-producing bacteria in human faecal samples and slurry cultures. Frontiers in Microbiology, 8, 1–11. doi:10.3389/fmicb.2017.01155.

Área SCI FI Q Citas “Microbiology” 4.019 Q2 (32/126) 15

. Artículo 2.- Vázquez, L., Flórez, A. B., Guadamuro, L. y Mayo, B. (2017). Effect of soy isoflavones on growth of representative bacterial species from the human gut. Nutrients, 9(7), 727. doi:10.3390/nu9070727.

Área SCI FI Q Citas “Nutrition & Dietetics” 4.196 Q1(18/83) 22

. Artículo 3.- Vázquez, L., Flórez, A. B., Redruello, B. y Mayo, B. (2020). Metabolism of soy isoflavones by intestinal bacteria: genome analysis of an Adlercreutzia equolifaciens strain that does not produce equol. Biomolecules, 10(6), 950. doi:10.3390/biom10060950.

Área SCI FI Q Citas “Biochemistry & Molecular 4.082 Q2 (98/297) 0 Biology” (2019)

. Artículo 4.- Vázquez, L., Flórez, A. B. y Mayo, B. (2020). Draft genome sequence of Adlercreutzia equolifaciens IPLA 37004, a human intestinal strain that does not produce equol from daidzein. Microbiology Resource Announcements, 9(8), e01537-19. doi:10.1128/MRA.01537-19.

Área SCI FI Q Citas No SCI - - 1

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. Artículo 5.- Vázquez, L., Flórez, A. B., Verbruggen, S., Redruello, B., Verhoeven, J., Venema, K. y Mayo, B. (2020). Modulation of equol production via different dietary regimens in an artificial model of the human colon. Journal of Functional Foods, 66, 103819. doi:10.1016/j.jff.2020.103819.

Área SCI FI Q Citas “Food science &Technology” 3.701 Q1(31/139) 1 (2019)

. Artículo 6.- Flórez, A. B., Vázquez, L., Rodríguez, J., Redruello, B. y Mayo, B. (2019). Transcriptional regulation of the equol biosynthesis gene cluster in Adlercreutzia equolifaciens DSM19450T. Nutrients, 11(5), 993. doi:10.3390/nu11050993.

Área SCI FI Q Citas “Nutrition & Dietetics” 4.546 Q1(17/89) 4

. Artículo 7.- Mayo, B., Vázquez, L. y Flórez, A. B. (2019). Equol: a bacterial metabolite from the daidzein isoflavone and its presumed beneficial health effects. Nutrients, 11(9), 2231. doi:10.3390/nu11092231.

Área SCI FI Q Citas “Nutrition & Dietetics” 4.546 Q1(17/89) 20

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