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Laboratory of Microbial Ecology and Technology (LabMET), Ghent University, Coupure Links 653, B-9000 Ghent, Belgium
2 To whom correspondence should be addressed. Email: willy.verstraete{at}UGent.be.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • equol • daidzein • gut bacteria • SHIME • soy germ
Dietary isoflavones, which exert a weak estrogenic activity, were investigated extensively for their potential role in preventing chronic disease, including cardiovascular disease, hormone-dependent cancer, osteoporosis, and postmenopausal syndrome (1–4). In soy and soy-based food products, the major sources of isoflavones, they occur mainly as unabsorbable and biologically inactive glycosides. Upon ingestion, a fraction is activated through deconjugation by intestinal hydrolases and absorbed (5). However, an important part of the glycosides reaches the colon directly or as glucuronic and sulfate conjugates after enterohepatic circulation (6,7). There, they are subjected to transformations by the intestinal microbiota that can lead to degradation or bioactivation. The importance, as well as the high interindividual variability of the gut microbial activity toward isoflavones with respect to their bioavailability and biological properties is well recognized (8). This is illustrated by the metabolic fate of daidzein (4',7-dihydroxy-isoflavone), one of the principal isoflavones, which can be microbiologically transformed into dihydrodaidzein (DHD),3 equol, and O-desmethylangolensin (O-DMA) (9).
Equol has received much attention recently because its biological activities differ from those of its precursor: it has higher estrogenicity (10), is a stronger antioxidant (11), and demonstrates antiandrogenic properties (12). Additionally, equol is easily absorbed through the colon wall, has a slower plasma clearance rate than daidzein, and is metabolically inert (13). However, only 30–50% of the Western population excrete significant amounts of equol (14,15). This fact was suggested as an explanation for the sometimes conflicting results obtained in the past from dietary intervention studies with isoflavones (13). Indeed, some reports suggested a lower disease risk for equol-producers than nonproducers (16–18). However, this is not fully proven and has been contradicted (19,20); more experimental data are necessary to explore the relation between equol production and health effects (21). Because equol was found to be formed exclusively by intestinal bacteria (22), its production in humans may depend on the presence of certain bacterial strains. A number of strains involved in daidzein metabolism were identified (23–25) and recently, we isolated a microbial consortium that catalyzes the conversion from daidzein into equol (26).
The composition and the activity of the gut microbiota are greatly predisposed by diet composition (27). Hence, it can be expected that dietary habits will influence equol production. However, previous reports have given conflicting results at times and have not yet led to the establishment of generic interpretations, if these exist (14,28–31). There is a growing interest in dietary applications that modulate equol production in humans. It was suggested that the use of functional foods containing certain bacteria could influence the equol production status of an individual (13). However, in 2 studies it was that reported the administration of probiotic lactic acid bacteria did not affect equol excretion in humans (32,33). Until the present, no food applications converting a nonequol-producer into a producer have been reported.
The research goal of this study was to investigate the influence of the supplementation of equol-producing bacteria on daidzein metabolism, the general composition and activity of a nonequol-producing intestinal microbial community. For this, we supplemented a microbial consortium (EPC4) efficiently transforming daidzein into equol, which we had isolated previously (26), to the Simulator of the Human Intestinal Microbial Ecosystem (SHIME) inoculated with fecal samples originating from nonequol-producing individuals.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Microbial strains and culture conditions. The bacterial strains used in this study were previously isolated from a human fecal sample at our laboratory (26). This stable mixed culture, which efficiently transforms daidzein into equol, consists of 4 dominant bacterial strains: Enterococcus faecium EPI1, Lactobacillus mucosae EPI2, Finegoldia magna EPI3, and a Veillonella sp.-related strain EP. The culture was grown anaerobically in brain heart infusion (Oxoid) supplemented with 0.5 g/L L-cystein (BHIa) as described previously (26).
Batch experiments (stomach and small intestine). To assess the resistance of the equol-producing capacity of EPC4 against digestion in the upper part of the digestive system, gastric and small-intestinal conditions were simulated in batch tests. Therefore, 10 mL of a 48-h old culture of EPC4 was added to an Erlenmeyer flask with 40 mL of SHIME suspension from the stomach and small intestine compartment, and incubated for 2 h at 37°C with shaking at 2.5 Hz. After incubation, the cells were centrifuged at 3000 x g and the pellet was washed twice with autoclaved saline (8.5 g/L NaCl). The washed pellet was then transferred to a penicillin flask containing autoclaved BHIa to which 200 µmol/L daidzein was supplemented and incubated as described above. At regular time intervals, samples were taken for HPLC analysis.
SHIME inoculum. Ethical approval for the experiments conducted in this study (protocol EC UZG 2004/044) was given by the Ethical Committee of the University Hospital of Ghent University. Fecal samples from 2 healthy women (age 23 and 25 y), who were known not to be equol-producers, were collected and used to inoculate the SHIME as described by De Boever et al. (35). The fecal microbial cultures obtained this way were denoted Fecal Inoculum (FI)-1 and FI-2. Part of the freshly voided samples was processed and assayed for daidzein metabolism as described earlier (26). Briefly, the microbial cultures of the fecal samples were washed after removal of the particulate matter and incubated under anaerobic conditions at 37°C in sterile BHIa to which 200 µmol/L daidzein was added. After 48 h, the samples were taken for analysis of daidzein and daidzein metabolites.
Experimental set-up.
Two identical SHIME-runs were performed with 2 different inocula: IF-1 and IF-2. A schematical representation of the experimental set-up is given in Figure 1. After reactor start-up, the system was allowed to stabilize for 3 wk with standard SHIME feed (37) supplemented with 7 g/L soy germ powder [SoylifeTMmicro25, Acatris Holding BV; the composition is described in De Boever et al. (35)] before the start of the experiment. The feed remained unchanged throughout the entire SHIME-run and contained 
175 mg/L isoflavones, of which 75 mg/L (175 µmol/L) had daidzein as aglycone. In this way, 41.3 µmol of daidzein equivalents were delivered daily to the SHIME. First, there was a 2-wk baseline period in which only the feed was dosed to the SHIME. This was followed by a 2-wk EPC4 inoculation period in which EPC4 was supplemented daily to the ascending colon compartment. Therefore, 10 mL of a 48-h-old culture of EPC4, whose OD at 590 nm was adapted to 0.5 with sterile brain heart infusion, corresponding to a total of 109 bacterial cells, was injected into reactor 3 of the SHIME. Finally, there was a postinoculation period in which no bacteria were supplemented and the SHIME was simply fed with standard SHIME feed, supplemented with 7 g/L soy germ powder. During the different phases, samples from the different colon reactors were taken at different time intervals and analyzed for daidzein and daidzein metabolites, short-chain fatty acids (SCFA) and DNA extraction. The total sampling volume that was retrieved from each compartment amounted to a maximum of 8 mL/d, which is <1.5% of the volume of each compartment. This did not affect the composition or activity of the SHIME.
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DNA extraction and RT-PCR. Total DNA extractions of 1 mL liquid SHIME sample were performed following the method described by Boon et al. (39). Using an ABI Prism SDS 7000 instrument (Applied Biosystems), general bacterial DNA as well as specific DNA from Lactobacillus spp., Bifidobacterium spp., Atopobium spp., and the Clostridium coccoides-Eubacterium rectale group were quantified by RT-PCR. Before amplification, DNA was diluted 10 times to dilute possible PCR-inhibiting compounds. The 16S rRNA genes for all members of the bacteria were amplified by PCR using the forward primer PRBA338f and the reverse primer P518r, following the protocol described by Boon et al. (39). Primer sets, annealing temperature, and Mg2+ concentration used to quantify the DNA of specific groups were described by Rinttilä et al. (40). Amplification was performed with a qPCRT core kit for SybrT Green I as described by the suppliers (Eurogentec) in MicroAmp Optical 96-well reaction plates with optical caps (PE Applied Biosystems), and the PCR protocol described by the last-mentioned group was adapted to obtain good amplification. The program started with 2 min at 50°C, followed by an initial denaturation step at 95°C for 5 min and 40 cycles of denaturation at 94°C for 20 s, primer annealing at specific temperature for 30 s, and primer extension at 60°C for 1 min. Standard curves were constructed with DNA from representative species of the different groups in a concentration range from 102 to 1010 DNA copies/µL, and DNA from at least 10 nontarget species was used as a negative control to test amplification specificity; all reactions proved to be specific for target species.
HPLC analysis. Two different HPLC methods were used in this study. For the extraction and analysis of daidzein, dihydrodaidzein, O-DMA, and equol the protocol described by Decroos et al. (26) was used. For the extraction of 6''-O-malonyl-daidzin, 6''-O-acetyl-daidzin, and daidzin, 1 mL of sample was diluted with ethanol to 70% (v:v) ethanol and then centrifuged for 5 min at 10000 x g. The supernatant was collected and evaporated completely under a flow of N2 at 37°C. The dried extract was dissolved in 1 mL ethanol:dimethyl sulfoxide (1:1), and samples were analyzed with the protocol described in De Boever et al. (41). An analytical standard for daidzin was kindly provided by Acatris Holding BV. The molar extinction coefficient that was determined for daidzin was used for quantification of malonyl- and acetyl-daidzin because these are very similar (42).
Statistical analysis. A comparison of the mean equol concentration at specific time points between 2 colon compartments within 1 SHIME-run was carried out using Student's t test. Data for daidzein metabolites, SCFA concentration, and microbial community structure were grouped per period and evaluated by 2-way ANOVA using period and colon compartment as main effects with testing for interactions (period x colon compartment). When significant effects or interactions were observed, means for different periods within 1 compartment and for different compartments within 1 period were compared using 1-way ANOVA and Duncan's post hoc test. For the daidzein metabolite concentrations, the criterion of homoscedasticy was not fulfilled. Therefore, data were evaluated using Weighted Least Square ANOVA, with a weight equal to the inverse of the variance assigned to each variable. When data were below the detection limit, we assigned a random value between 0 and the detection limit, which was 0.85, 0.96, 1.2, and 0.74 µmol/L for daidzein, DHD, equol, and O-DMA, respectively. Differences were considered significant at P < 0.05. Values in the text are means ± SD. All statistical analyses were performed using SPSS 12.0 for Windows software.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Daidzein metabolism of the fecal inocula in batch tests. Before the start-up of the SHIME, the capacity of the 2 fecal samples (further used to inoculate the SHIME) to metabolize daidzein was tested in batch (Table 1). FI-1 produced DHD and O-DMA, whereas FI-2 produced only DHD. FI-2 degraded more daidzein than FI-1, i.e., 33 ± 3 and 45 ± 5% of the originally added amount of daidzein was transformed for FI-1 and FI-2, respectively.
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1 d compared with the transverse colon compartment. When the equol concentrations at each time point were compared between the transverse and descending colon compartment within the same SHIME-run, equol concentrations were significantly lower (P 
0.035) in the descending colon compartment from d 20–23 for both SHIME-runs. At every other time point, equol concentrations did not differ (P 
0.087). Coinciding with the appearance of equol, both daidzein and DHD concentrations decreased and remained negligible until d 42 for both experiments.
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Daidzein metabolism over the entire gastrointestinal tract. The sum of all of the daidzein-related compounds did not differ over the whole simulated gastrointestinal tract for both the runs with FI-1 (P = 0.06, 158 ± 17 µmol/L) and FI-2 (P = 0.19, 152 ± 12 µmol/L). In both SHIME-runs, a shift occurred from the malonyl- and acetyl-glycosylated and glycosylated forms in the feed toward the aglycone in the duodenum and ascending colon compartments and further to O-DMA and equol in the distal colon parts. In the descending colon, equol accounted for 79 ± 4% and O-DMA for 19 ± 4% (on a molar-basis) of the daidzein equivalents administered to the SHIME through the feed in the run with FI-1. In the run with FI-2, 99 ± 6% of the administered daidzein was found back as equol in the descending colon.
Production of SCFA. The concentrations of SCFA were measured during the different periods of the SHIME-runs in the different colon compartments of the SHIME (Table 3). The most important feature that was noted was the significant increase in butyrate production in both SHIME-runs in all of the colon vessels during the EPC4 inoculation period, which lasted into the postinoculation period. In the run with FI-2, this was associated with a decrease in propionate concentrations in all colon compartments.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The results presented here are a first "proof of principle" that supplementation with equol-producing bacteria can constitute a novel way to induce or enhance equol production in situ. For a number of reasons, it is was suggested that the presence of equol is an important factor in the positive clinical effects of a diet rich in soy (13). Although this is not yet fully supported by experimental data, and the health benefits of equol production remain subject to further investigation (21), there is much interest in food applications that modulate the intestinal production of equol. Here, we showed that supplementation with EPC4, an equol-producing bacterial consortium we isolated from a human fecal sample, can convert a nonequol-producing into an equol-producing intestinal microbial community in vitro. This indicates that the equol-producing bacteria from EPC4 are able to survive in a complex microbial community and beyond that, maintain their metabolic activity. They were able to proliferate efficiently in the simulated colon because equol production was maintained up to 2 wk after ending the treatment.
Equol was detected only in the transverse and descending colon compartments. The net production was limited to the transverse colon compartment, but it cannot be excluded that this could also take place in the descending colon compartment because all of the daidzein was already consumed in the former compartment. This was confirmed in batch tests with suspensions drawn from the descending colon compartment to which daidzein was supplemented and in which equol was produced after 24 h of anaerobic incubation (data not shown). The equol present in the descending colon compartment at concentrations similar to those in the previous reactor was the result of hydraulic overflow and further substantiated the resistance of equol to further bacterial breakdown (13,26). The manifestation of equol production not earlier than the transverse colon is probably due to the environmental conditions that are necessary for growth and metabolic activity of equol-producing bacteria. The most plausible determining factor would be the redox potential which decreases progressing in the colon to –250 mV in vivo (43) and from –190 mV in the ascending colon compartment to –240 mV in the descending colon compartment in the SHIME (this study). The strictly anaerobic character of equol-producing bacteria was suggested earlier (44), and a recently reported equol-producing isolate was also strictly anaerobic (25). The fact that equol was produced only in the distal colon compartments is in good agreement with in vivo data on plasma concentrations of equol. Zubik et al. (45) found that peak plasma concentrations appeared 24 h postprandially after a single soy challenge; this is the time in vivo after ingestion in which food reaches the distal parts of the colon (46). In addition, data on peaks in urinary excretion of equol suggest formation of the compound in more distal parts of the colon (47,48).
The inter-regional differences in colonic environmental conditions were well reflected in the daidzein metabolism in both SHIME-runs. In the proximal colon compartment (ascending colon), daidzein metabolism was limited. This part of the colon is characterized by a higher redox potential, higher carbohydrate concentration, and lower pH than the distal parts (27). The high redox potential could be a limiting factor for reductive metabolism, and carbohydrates were already shown to inhibit daidzein degradation (49) and equol production (26,50). Like equol, O-DMA was produced only in the distal colon compartments. Because all bacterial strains able to form O-DMA isolated to date are anaerobic (51,52), this is probably due to the lower redox potential in distal colon regions.
Thus far, bacteria that target specific transformations of certain compounds related to beneficial health effects have not been considered for human consumption. However, because knowledge about the complex processes in the colon is increasing, the latter could be next-generation bacterial supplements. Because EPC4 could not maintain its equol-producing capacity under the acidic and biliary stress conditions in the stomach and small intestine, it could be limited in its potential application as an oral supplement. However, this could be circumvented by specific encapsulation of the strains, releasing them not earlier than the colon (53). Although all of the strains present in EPC4 are normal inhabitants of a healthy intestinal microbiota, the safety of supplementing these bacteria, which are not generally recognized as safe (GRAS) to humans, must be investigated thoroughly. As far as it can be derived from the data obtained in this work, administration of EPC4 did not have adverse effects on the general composition and activity of the microbial community. On the contrary, inoculation with EPC4 increased the Lactobacillus population, probably as a consequence of the presence of Lactobacillus mucosae EP1 in EPC4, and also of Enterococcus faecium EP2, which can lower colonic pH through the production of lactic acid, thus favoring the environmental conditions for lactic acid bacteria (54). Lactobacilli have positive effects on colonic health (55). Furthermore, we observed an increase in butyrate production upon inoculation with EPC4. The latter compound plays an important role in cell differentiation, is an energy source for colonocytes, and can induce apoptosis of cancerous cells in vitro, thus inhibiting colon cancer development (56).
In conclusion, we found that administration of equol-producing bacteria to a dynamic in vitro model of the gastrointestinal tract, inoculated with a nonequol-producing fecal sample, resulted in the formation of equol in the distal colon parts. This effect was maintained beyond the period of inoculum administration. Although further mechanistic studies and experiments with in vivo models are necessary to explore the efficacy and safety of EPC4, these results comprise a first validation of the concept that equol-producing bacteria can be used as a novel means for the induction or enhancement of equol production in situ. Furthermore, the SHIME was an excellent tool for the study of the intestinal metabolism of daidzein, revealing interregional differences in colonic biotransformation processes that can be explicative for in vivo data on urinary and plasma concentrations of daidzein metabolites. This suggests that the SHIME can be used successfully for mechanistic studies on the intestinal bioactivation or degradation of phytoestrogens and similar compounds.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BHIa: brain heart infusion supplemented with 0.5 g/L L-cystein; DHD, dihydrodaidzein; FI, fecal inoculum; GRAS: Generally Recognized As Safe; O-DMA: O-desmethylangolensin; SCFA, short-chain fatty acids.
Manuscript received 3 November 2005. Initial review completed 9 December 2005. Revision accepted 17 January 2006.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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