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      Equol status and changes in fecal microbiota in menopausal women receiving long-term treatment for menopause symptoms with a soy-isoflavone concentrate

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          Abstract

          The knowledge regarding the intestinal microbial types involved in isoflavone bioavailability and metabolism is still limited. The present work reports the influence of a treatment with isoflavones for 6 months on the fecal bacterial communities of 16 menopausal women, as determined by culturing and culture-independent microbial techniques. Changes in fecal communities were analyzed with respect to the women’s equol-producing phenotype. Compared to baseline, at 1 and 3 months the counts for all microbial populations in the feces of equol-producing women had increased strongly. In contrast, among the non-producers, the counts for all microbial populations at 1 month were similar to those at baseline, and decreased significantly by 3 and 6 months. Following isoflavone intake, major bands in the denaturing gradient gel electrophoresis (DGGE) profiles appeared and disappeared, suggesting important changes in majority populations. In some women, increases were seen in the intensity of specific DGGE bands corresponding to microorganisms known to be involved in the metabolism of dietary phytoestrogens ( Lactonifactor longoviformis, Faecalibacterium prausnitzii, Bifidobacterium sp., Ruminococcus sp.). Real-Time quantitative PCR revealed that the Clostridium leptum and C. coccoides populations increased in equol producers, while those of bifidobacteria and enterobacteria decreased, and vice versa in the non-producers. Finally, the Atopobium population increased in both groups, but especially in the non-producers at three months. As the main findings of this study, (i) variations in the microbial communities over the 6-month period of isoflavone supplementation were large; (ii) no changes in the fecal microbial populations that were convincingly treatment-specific were seen; and (iii) the production of equol did not appear to be associated with the presence of, or increase in the population of, any of the majority bacterial types analyzed.

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          Use of 16S rRNA gene-targeted group-specific primers for real-time PCR analysis of predominant bacteria in human feces.

          16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium, and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68:5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log10 10.3 +/- 0.3 cells per g [wet weight] [average +/- standard deviation]), followed by the C. leptum subgroup (log10 9.9 +/- 0.7 cells per g [wet weight]), the B. fragilis group (log10 9.9 +/- 0.3 cells per g [wet weight]), Bifidobacterium (log10 9.4 +/- 0.7 cells per g [wet weight]), and the Atopobium cluster (log10 9.3 +/- 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log10 9.7 +/- 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.
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            Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces.

            For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.
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              Antibacterial, antifungal, and antiviral activities of some flavonoids.

              Antibacterial and antifungal activities of six plant-derived flavonoids representing two different structural groups were evaluated against standard strains of Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumoniae, Acinetobacter baumannii, Staphylococcus aureus, Enterococcus faecalis, Bacillus subtilis and their drug-resistant isolates, as well as fungi (Candida albicans, C. krusei) using the microdilution broth method. Herpes simplex virus Type-1 and Parainfluenza-3 virus were employed for antiviral assessment of the flavonoids using Madin-Darby bovine kidney and Vero cell lines. Ampicillin, gentamycin, ofloxacin, levofloxacin, fluconazole, ketoconazole, acyclovir, and oseltamivir were used as the control agents. All tested compounds (32-128 microg/ml) showed strong antimicrobial and antifungal activities against isolated strains of P. aeruginosa, A. baumanni, S. aureus, and C. krusei. Rutin, 5,7-dimethoxyflavanone-4'-O-beta-D-glucopyranoside and 5,7,3'-trihydroxy-flavanone-4'-O-beta-D-glucopyranoside (0.2-0.05 microg/ml) were active against PI-3, while 5,7-dimethoxyflavanone-4'-O-[2''-O-(5'''-O-trans-cinnamoyl)-beta-D-apiofuranosyl]-beta-D-glucopyranoside (0.16-0.2 microg/ml) inhibited potently HSV-1. Copyright 2009 Elsevier GmbH. All rights reserved.
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                Author and article information

                Contributors
                Journal
                Front Microbiol
                Front Microbiol
                Front. Microbiol.
                Frontiers in Microbiology
                Frontiers Media S.A.
                1664-302X
                05 August 2015
                2015
                : 6
                : 777
                Affiliations
                [1] 1Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones Científicas Villaviciosa, Spain
                [2] 2Servicios Científico-Técnicos, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones Científicas Villaviciosa, Spain
                [3] 3Servicio de Digestivo, Hospital de Cabueñes Gijón, Spain
                [4] 4Consorcio de Apoyo a la Investigación Biomédica en Red, Hospital Universitario Central de Asturias Oviedo, Spain
                [5] 5Facultad de Ciencias de la Educación, Universidad Autónoma de Chile Santiago, Chile
                Author notes

                Edited by: Javier Carballo, University of Vigo, Spain

                Reviewed by: Analia Graciela Abraham, Centro de Investigacion y Desarrollo en Criotecnologia de Alimentos, Argentina; Amit Kumar Tyagi, The University of Texas MD Anderson Cancer Center, USA

                *Correspondence: Baltasar Mayo, Departmento de Microbiologia y Bioquimica, Instituto de Productos Lácteos de Asturias – Consejo Superior de Investigaciones Científicas, Paseo Río Linares s/n, 33300-Villaviciosa, Asturias, Spain, baltasar.mayo@ 123456ipla.csic.es

                These authors have contributed equally to this work.

                This article was submitted to Food Microbiology, a section of the journal Frontiers in Microbiology

                Article
                10.3389/fmicb.2015.00777
                4525046
                26300856
                72e1995b-3aa0-411a-be46-a63fd9d521f6
                Copyright © 2015 Guadamuro, Delgado, Redruello, Flórez, Suárez, Martínez-Camblor and Mayo.

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                : 19 May 2015
                : 15 July 2015
                Page count
                Figures: 1, Tables: 4, Equations: 0, References: 44, Pages: 10, Words: 0
                Funding
                Funded by: Spanish Ministry of Economy and Competitiveness
                Award ID: AGL2011-24300
                Award ID: AGL-2014-57820-R
                Funded by: MINECO
                Award ID: BES-2012-062502
                Funded by: CSIC JAE-Doc Program
                Categories
                Microbiology
                Original Research

                Microbiology & Virology
                soy isoflavone,equol,intestinal microbiology,fecal microbiota,menopause,probiotics

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