Metabolism of flaxseed lignans in the rumen and its impact on ruminal metabolism and flora

Animal Feed Science and Technology 150 (2009) 18–26

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Animal Feed Science and Technology journal homepage: www.elsevier.com/locate/anifeedsci

Metabolism of flaxseed lignans in the rumen and its impact on ruminal metabolism and flora Wei Zhou a, Guojie Wang a, Zhengkang Han a,∗, Wen Yao b, Weiyun Zhu b a

Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture of China, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China College of Animal Science and Technology, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China

b

a r t i c l e

i n f o

Article history: Received 19 January 2008 Received in revised form 31 May 2008 Accepted 17 July 2008 Keywords: Flaxseed lignans Mammalian lignans Enterolactone Enterodiol Ruminal metabolism Goat Denaturing gradient gel electrophoresis

a b s t r a c t Lignans belong to a group of phytoestrogens found in many edible plants. They can be converted to mammalian lignans, namely enterodiol (END) and enterolactone (ENL), in non-ruminant animals. Previous studies have shown that lignans may possess estrogen-like biological properties and can interact with various enzymes and proteins in rats and human. Similar reactions in ruminants have not yet been demonstrated. The metabolism of lignans in the rumen and its impact on ruminal metabolism was therefore investigated in 6 Huainan goats fitted with permanent rumen fistulae and temporary jugular catheter which were supplied with flaxseed lignan (secoisolariciresinol diglucoside, SDG) via rumen fistulae. Low concentrations of END (0.017; S.D. = 0.024 mg/mL) and ENL (0.007; S.D. = 0.013 mg/mL) were detected in ruminal fluid of the goats fed the basal diet but concentration of SDG or mammalian lignans in the serum were below the detection level. Following SDG administration at 1 mg/kg body weight for 14 d, the concentrations of the three lignans increased (P<0.01). Concomitantly, the pH (P<0.05) and ammonia–nitrogen concentration (P<0.01) of ruminal fluid were decreased, while the concentration of microbial crude protein (P<0.05) and total volatile fatty acids (P<0.01) increased.

Abbreviations: SDG, secoisolariciresinol diglucoside; END, enterodiol; ENL, enterolactone; BD, basal diet; DGGE, denaturing gradient gel electrophoresis; VFA, volatile fatty acids; TDHA, total dehydrogenase activity; MCP, microbial crude protein; NH3 –N, ammonia–N; (A + B):P, (acetate + butyrate) to propionate ratio. ∗ Corresponding author. Tel.: +86 25 84396224; fax: +86 25 84398669. E-mail address: [email protected] (Z. Han). 0377-8401/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.anifeedsci.2008.07.006

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Based on denaturing gradient gel electrophoresis profiles, SDG supplementation showed an effect on the rumen microbial profile. The results demonstrated that ruminal microorganisms efficiently converted SDG to END and ENL, and that SDG supplementation may improve ruminal metabolism of carbohydrates and nitrogenous compounds and influence the bacterial composition in the rumen of goats. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Phytoestrogens are estrogen-like compounds found in plants, comprised of two families, namely isoflavones and lignans. Lignans, ubiquitous in many edible plants, are the main source of dietary phytoestrogens. Flaxseed is the richest known source of mammalian lignan precursors, especially secoisolariciresinol diglucoside (SDG); SDG content varies between 11.7 and 24.1 mg/g in defatted flour and between 6.1 and 13.3 mg/g of whole flaxseed (Johnsson et al., 2000). Dietary lignans, particularly SDG, are of interest because they may help to prevent breast and colon cancers (Thompson, 1998), atherosclerosis (Prasad, 1999), and diabetes (Prasad et al., 2000). Previous studies with non-ruminant animals have demonstrated that plant lignans (SDG) per se are devoid of any biological properties; only the mammalian lignans enterodiol (END) and enterolactone (ENL), produced from SDG by intestinal bacteria, possess biological properties such as estrogen agonism and antagonism as well as antioxidation and enzyme inhibition (Clavel et al., 2005). However, bacterial activation (conversion) of SDG in the rumen and the impact of SDG or its metabolites – END and ENL – on microorganisms remain poorly understood. Studies conducted in the 1980s showed that long-term consumption of large amounts of legumes could detrimentally affect the fertility of ruminants, causing the so-called “clover disease” due to the isoflavones released from legumes (Van Soest, 1994). As reviewed by Zhengkang et al. (2006), injection of daidzein, one of the main isoflavones, via duodenum cannulae could increase serum levels of testosterone, concentration of bacterial protein, ammonia nitrogen (NH3 –N), and total volatile fatty acids (TVFA) of rumen fluid. Because both isoflavones and lignans have a phenolic group, similar to estrogenic steroids, they may also have similar physiological actions on rumen metabolism. In order to confirm this hypothesis and solve the following questions (Is SDG metabolized to END or ENL in the rumen? If so, are these products absorbed into the circulation? Further, how do SDG and/or the enterolignans alter ruminal metabolism?), we investigated the metabolism of lignans in the rumen and its impact on ruminal metabolism and flora. Therefore, our goal was to understand fully the interactions between rumen metabolism and lignans, and to observe the influence of gender on rumen metabolism. 2. Materials and methods 2.1. Animals and management Six healthy Huainan goats fitted with ruminal cannulae were housed individually in well-ventilated cement-floored pens and maintained under strict hygienic conditions. Goats were fed a diet of Chinese wildrye hay (1 kg, Leymus chinensis [Trin.] Tzvel.) and concentrate (200 g) twice daily at 08:00 am and 18:00. Concentrate was ground corn grain and soybean meal (7:3, dry matter basis). Goats had free access to fresh water. 2.2. Experimental design and sampling In a crossover study with 2 periods, goats were assigned randomly to 2 groups of 3 animals each (2 males and 1 female) on the basis of body weight (20 ± 2.5 kg). Each period lasted 4 weeks, with 14 d for either adaptation to or washout of SDG, followed by 2 weeks of sampling. Twice a day during

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the treatment period, 1 mg/kg body weight SDG (Chromadex, USA) was infused into the rumen of the goats via the fistulae. On days 14, 21, and 28, samples of ruminal contents obtained via ruminal cannula and 2 mL blood (collected through a catheter in the jugular vein) were obtained from each goat at 07:00 am, and every 2 h thereafter for the next 22 h. The pH of the ruminal fluid and the total dehydrogenase activity (TDHA) were determined using fresh ruminal samples; blood samples were centrifuged (1000 × g for 15 min) to obtain sera that were stored at −20 ◦ C until analyzed. Following procedures of Konstantinov et al. (2003), 3 mL of whole ruminal contents to be used later for DNA extraction and analysis with a denaturing gradient gel were stored separately and maintained at −20 ◦ C for 2 months. Other rumen samples were filtered through 4 layers of cheesecloth. A 10 mL aliquot of the filtered ruminal fluid was acidified with 1 mL of 6 N HCl and stored in a freezer (−20 ◦ C) for NH3 –N analysis. Further, 2 mL of freshly prepared 25% (250 mL/L) metaphosphoric acid was added to 8 mL of the filtered ruminal fluid. These samples were centrifuged (17,000 × g for 10 min) with the supernatant stored at −20 ◦ C for later analysis of volatile fatty acids (VFAs). 2.3. Chemical analysis The concentrations of SDG and the 2 mammalian lignans in both the ruminal fluid and serum were measured using high-performance liquid chromatography (HPLC) (Agilent 1100 Series; USA). Based on the respective authentic standards (SDG: Chromadex, USA; END and ENL: Sigma, USA) as described by Morton et al. (1997). Since Adlercreutz et al. (1995) found that 92% of END and 98% of ENL exist as glucuronide conjugates in the serum, the procedure for the analysis of serum END and ENL included a NaOH hydrolysis step (1 mol/L NaOH at 20 ◦ C for 48 h). NH3 –N was measured using the indophenol method (Weatherburn, 1967). The VFAs in the ruminal fluid were measured by using a capillary column gas chromatograph (GC-14A; Shimadzu, Japan) (Mao et al., 2007). The microbial crude protein (MCP) content was determined by the purine-based method of Zinn and Owens (1986), based on the ratio of purines to nitrogen in the isolated microbes. TDHA was analyzed following the method described by Dror et al. (1969). 2.4. Denaturing gradient gel electrophoresis (DGGE) of partial 16S rRNA genes The rumen content samples obtained from each goat at day 14 (11:00 h) of the control period and days 7 and 14 (11:00 h) of the treatment period were homogenized, and 1 g aliquots were used for DNA extraction. DNA was extracted using the bead beating method with a mini bead beater (Biospec Products, USA) followed by phenol–chloroform extraction (Zoetendal et al., 1998). The solution was then precipitated with ethanol, and pellets were suspended in 50 ␮L of TE. The V6–V8 regions of bacterial 16S rDNA were amplified by polymerase chain reaction (PCR) using the following primers: U968-GC (5 -CGC CCG GGG CGC GCC CCG GGC GGG GCG GGG GCA CGG GGG GAA CGC GAA GAA CCT TAC-3 ; the underlined sequence corresponds to the GC clamp) and L1401 (5 -CGG TGT GTA CAA GAC CC-3 ) (Nübel et al., 1996). PCR was performed with a Taq DNA polymerase kit (Promega, USA). The samples were amplified by using a T1 Whatman Biometra (Göttingen, Germany) with the following conditions: initial denaturation at 94 ◦ C for 5 min; 35 amplification cycles with 30 s of denaturation at 94 ◦ C, 20 s of annealing at 56 ◦ C, and 40 s of extension at 68 ◦ C; and a final extension at 68 ◦ C for 7 min. Aliquots of 5 ␮L were electrophoretically analyzed on an agarose gel (12 g/L) containing ethidium bromide in order to examine the sizes and amounts of the amplicons. The amplicons of the V6–V8 regions of 16S rDNA were used for sequence-specific separation by DGGE according to the specifications of Muyzer et al. (1993) by using the Dcode DGGE system (Bio-Rad, USA). DGGE was performed on polyacrylamide gels (80 g/L) containing 37.5:1 acrylamide–biacrylamide and a denaturing gradient of urea (380–530 g/L). The electrophoresis was initiated by pre-running for 10 min at a voltage of 200 V, and it was subsequently run at a fixed voltage of 85 V for 12 h at 60 ◦ C. After completion of electrophoresis, the gels were stained with AgNO3 (Sanguinetti et al., 1994). They were then scanned using a calibrated densitometer (GS 800; Bio-Rad, USA).

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As described by Collier et al. (2003), when SDG-dependent differences were observed in the PCRDGGE banding profiles, the distinguished bands were excised, reamplified as described for PCR-DGGE, and sequenced on the ABI PRISM 377 sequencer (PerkinElmer) at Invitrogen, China). These sequences then were compared with those available on public databases by basic local alignment search tool (BLAST) analysis of sequences from the Ribosomal Database (http://www.ncbi.nlm.nih.gov/BLAST/). 2.5. Statistical analyses In order to control the influence of factors such as circadian rhythm, sampling time, feeding time, water intake, etc., for metabolic variables, such as MCP level, all samples were analyzed separately. Mean levels of each metabolism parameter were then calculated for each day and goat. Differences due to treatment, gender and the interactions between them were tested for significance using the two-way ANOVA by SPSS system (V11.5). Data were expressed as means and standard deviations (S.D.). The statistical model used was as follows: Y =  + St + Gd+ (S*G)td + εtdk. Where Y is the parameter to be tested,  is the overall mean, St is the effect of SDG treatment, Gd is the effect of gender, (S*G)td denotes the interaction between SDG treatment and gender, εtdk is the error term. Similarity indices were calculated for pairs of DGGE profiles from the densitometric curves of the scanned DGGE profiles by using the Molecular Analyst 1.61 software (Bio-Rad, USA). As a parameter for the structural diversity of the microbial community, the Shannon index of general diversity HP (Shannon and Weaver, 1963; Eichner et al., 1999; McCracken et al., 2001) was calculated for the bands in a lane by using the following formula: HP = −˙Pi log Pi. HP was calculated on the basis of the bands on the gel tracks that were applied for the generation of the dendrograms by using the intensities of the bands as determined by the peak height in the densitometric curves. The importance probability Pi was calculated as Pi = ni/HP, where ni is the height of a peak and HP is the sum of all peak heights in the densitometric curve. 3. Results 3.1. Lignan concentration in the ruminal fluid and serum In this study, the levels of mammalian lignans in the serum and ruminal fluid of the 6 goats were used to assess the bacterial activation and absorption of SDG. As shown in Fig. 1A and D, there were low levels of END (0.017 ± 0.024 mg/mL) and ENL (0.007 ± 0.013 mg/mL) in the ruminal fluid under the basal diet condition. This may indicate that ruminal microorganisms efficiently converted SDG to enterolignans; however, those of SDG or mammalian lignans in the serum were under the detected level. As shown in Fig. 1B and C, after 14 d of exposure to SDG, the levels of mammalian lignans and SDG in both the ruminal fluid and serum increased (P<0.01); meanwhile, the levels of ENL in the 2 biofluids were nearly twice those of END. 3.2. Effects of SDG on ruminal metabolism Effects of SDG supplementation on ruminal fermentation are shown in Table 1. Although large gender-related differences were observed, impacts of SDG addition were similar between the different genders for all of the metabolism parameters. In the 6 goats, after 14 d of exposure to SDG, the pH value of the ruminal fluid was decreased (P<0.05), the TVFA level (P<0.01) and the concentrations of individual VFAs were increased (P<0.01); however, no remarkable change was observed in the TDHA and the ratio of individual VFAs except butyrate. Rumen bacterial protein synthesis also was positively affected; the NH3 –N concentration was reduced (P<0.01), and the MCP concentration in ruminal fluid increased (P<0.05). 3.3. Effects of SDG on ruminal microorganisms The impact of SDG supplementation on the microbial profile was investigated by DGGE of V6–V8 amplicons. A representative DGGE analysis of the PCR fragments generated with primers

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Fig. 1. Concentrations of secoisolariciresinol diglucoside (SDG), enterodiol (END), and enterolactone (ENL) in the ruminal fluid and serum. (A) Levels of SDG and the 2 mammalian lignans in the ruminal fluid under the basal diet (BD) condition. (B) Levels of SDG and the 2 lignans in the ruminal fluid after 14-d exposure to SDG. (C) Levels of SDG and 2 lignans in the serum after 14-d exposure to SDG. (D) Mean concentrations of SDG and the 2 lignans in the different periods and biofluids.

968-GC and 1401r is shown in Fig. 2. Although most of the DGGE bands were common to both the control and SDG-treated rumen samples, some bands became more intense while others disappeared after the addition of SDG. For example, the band indicated as 1 was common to most rumen samples with BD or SDG supplementation at 7 d, but it disappeared after exposure to SDG for 14 d. Band 3 was observed only in the BD rumen samples, while the intensity of band 2 tended to increase after SDG supplementation. The results suggested that these bands responded to the addition of SDG. In order to identify the 3 most predominant bacteria, 16S

Table 1 Impact of secoisolariciresinol diglucoside (SDG) on ruminal metabolism Control

SDG treatment

Males pH TDHA NH3 –N MCP TVFA A P B (A + B)/P

6.33 0.99 1.37 1.16 10.17 5.77 2.80 1.60 2.54

Females ± ± ± ± ± ± ± ± ±

0.07 0.12 0.14 0.17 0.73 0.94 0.31 0.12 0.10

6.13 0.46 1.93 0.60 18.61 8.39 6.38 3.84 1.92

± ± ± ± ± ± ± ± ±

Males 0.02 0.04 0.18 0.15 1.25 1.08 0.37 0.06 0.14

6.18 1.24 1.05 2.01 18.25 9.84 4.70 3.71 2.80

Main effect Females

± ± ± ± ± ± ± ± ±

0.02 0.120 0.05 0.35 1.03 1.35 0.43 0.37 0.04

6.07 0.68 1.05 0.97 23.27 11.23 7.37 4.67 2.16

± ± ± ± ± ± ± ± ±

0.04 0.05 0.03 0.20 1.44 0.86 0.32 0.39 0.03

SDG

Gender

SDG*Gender

0.022 0.931 <0.001 0.013 <0.001 0.001 0.001 0.003 0.181

0.001 0.607 0.006 0.003 <0.001 0.001 0.004 0.017 0.027

0.042 0.699 0.030 0.048 <0.001 0.002 0.005 0.002 0.985

TDHA: total dehydrogenase activity (U/100 mL ruminal fluid); NH3 –N: ammonia–N (mg/mL); MCP: microbial crude protein (mg/mL); TVFA: total volatile fatty acids (mmol/L); A: acetate (mmol/L); P: Propionate (mmol/L); B: butyrate (mmol/L); (A + B)/P: (acetate + butyrate) to propionate ratio.

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Fig. 2. DGGE of PCR products of the V6–V8 regions of 16S rDNA from rumen content samples from goats. (a) Goats A, B, C, D, E and F on basal diet (BD). (b) The 6 male goats on the BD with SDG at 14 d. (c) The 6 male goats on the BD with SDG at 7 d. Two bands, indicated as 1 and 3, are common to most rumen content samples with BD, but they disappear after 14 d of SDG treatment. The band indicated as 2 is only common to rumen contents samples after 14 d of SDG treatment. SDG: secoisolariciresinol diglucoside.

rDNA was sequenced. Band 2 was most closely related to Ruminococcus gnavus (R. gnavus), with 98% similarity. Although the similarity level of the sequences corresponding to bands 3 and 1 with the database sequences was in the range of 88–91%, their closest relatives are unidentified bacteria. In order to control individual variation, similarity comparisons between BD and SDG treatment at day 14 were individually calculated for each goat. The band similarity coefficient in goats A, B, C, D, E, and F were 75.0, 56.8, 71.1, 60.5, 62.9, and 74.7%, respectively. To further examine the effect of SDG, we analyzed the number of predominant bands in the DGGE profiles and their Shannon index of general diversity (Fig. 3). The total number of bands for the rumen samples increased in a time-dependent manner and was the highest (P<0.05) after 14-d SDG exposure. Even though the Shannon index of diversity HP values were identical after 7 and 14 d of SDG treatment, i.e., 1.390, the standard deviations tended to be smaller after 14 d of exposure to SDG (P=0.045).

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Fig. 3. Analysis of denaturing gradient gel electrophoresis (DGGE) banding patterns in SDG-treated or non-treated goats. (A) Total number of DGGE bands. (B) Diversity index of DGGE profiles. Data are expressed as mean ± standard deviations. * P<0.05 compared with the control.

4. Discussion 4.1. Transformation and absorption of SDG in the rumen The transformation and absorption of plant lignans in humans and rats have been studied since ENL and END were first isolated from the urine of humans in 1980 (Wang, 2002). The possible metabolic fate of lignans in monogastric animals appears to include several steps. First, after deglycosylation, SDG is transformed to secoisolariciresinol (SECO); SECO then undergoes demethylation and dehydroxylation catalyzed by intestinal bacteria, whereby it is converted to END, which could further dehydroxylated to yield ENL. Once formed, END and ENL enter the enterohepatic circulation and are eventually excreted in the urine mainly as glucuronide conjugates (Kuijsten et al., 2005). In our study, the increase in mammalian lignans (END and ENL) observed both in the rumen and circulation following SDG supplementation suggests that ruminal microorganisms efficiently convert SDG to enterolignans. Moreover, after 14-d exposure to SDG, the area under the curve for serum ENL was found to be nearly twice that for serum END; this may indicate that ENL is the main circulatory lignan in goats. Bowey et al. (2003) and Knust et al. (2006) have demonstrated that when SDG was administered to humans or human flora-associated rats, a large amount of ENL was formed; in contrast, Tan et al. (2004) found that in rats, the concentration of urinary END was nearly 10 times that of urinary ENL. The lignan profiles therefore may depend on the composition of the dominant intestinal microbiota in different animals. As shown in Fig. 1B, the SDG concentration in the ruminal fluid increased slowly after SDG infusion. One possible explanation for this observation was that SDG was released gradually because SDG present in flaxseed is part of a macromolecule in which it is connected through the linker molecule hydroxymethyl glutaric acid (HMGA). When the data corresponding to days 21 and 28 were compared with that corresponding to day 14 of sampling, the levels of both SDG and the 2 mammalian lignans in the rumen and circulation were similar; this may indicate that after 14 d of exposure to SDG, the transformation and absorption of SDG in goats had reached a relatively high level and stabilized. 4.2. Effects of SDG on ruminal metabolism and microorganisms In ruminal fluid, the NH3 –N concentration declined during SDG administration, but the MCP level increased remarkably; this indicates that SDG may promote the utilization of nonprotein nitrogen. Positive effects of daidzein on MCP in rumen and protein synthesis of muscle cells in rats were reported in a review by Zhengkang et al. (2006). We, therefore, believe that the increased MCP concentration and the decreased NH3 –N level in the present study may have been caused by SDG and/or the two mammalian lignans. Simultaneously, the TVFA level and the concentrations of individual VFAs were increased (P<0.01) in the ruminal fluid. SDG may facilitate the anabolic metabolism of carbohydrates and alter the fermentation pattern. These impacts were confirmed by an in vitro study carried out by Wang et al. (2007). Mao et al. (2007) demonstrated that daidzein, another important phytoestrogen,

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could dramatically alter the acetate: propionate ratio and reduce the concentration of NH3 –N in the rumen, while MCP tended to increase. The causal mechanism appears to be the direct effect of daidzein on rumen microorganisms. Thus, isoflavones and lignans may have similar physiological functions in ruminal metabolism due to their common estrogen-like activities. Although previous studies with non-ruminant animals have reported that plant lignans per se are devoid of any biological action (Clavel et al., 2005), elucidation of biological properties with regard to ruminal metabolism requires further studies. The relationship between SDG and the intestinal flora present in humans and rats has been studied widely, but these studies have focused mainly on the types of bacteria involved in the conversion of dietary SDG. The impact of SDG or its metabolites, namely, END and ENL, on microorganisms and the underlying mechanisms remain poorly understood, particularly for ruminants. To examine the effect of SDG on microbial composition, we adopted a PCR-DGGE approach combined with sequence analysis in this study to monitor the alterations in the rumen bacterial community of goats. The results demonstrated that the composition of the bacterial community in the rumen content samples different between goats treated with SDG and the non-treated goats. SDG addition increased the number of predominant bands (P<0.05) and the Shannon index of general diversity (P<0.05) in the DGGE profiles. According to Stackebrandt and Goebel (1994), the similarity threshold at which species are generally considered identical is 97%. In this study, the sequence marked as 2 that increased was identified as belonging to R. gnavus. Since ␤-glucuronidase activity of R. gnavus E1 has been demonstrated by Beaud et al. (2005), 1 mol SDG may supply R. gnavus with 2 mol glycosides. Meanwhile, Clavel et al. (2007) reported that Ruminococcus productus was involved in the O-demethylation of SCEO to END. This may indicate that SDG stimulates the growth of R. gnavus with SDG as the substrate. Although the other clones indicated as 3 and 1 have a similarity level in the range 88–91% with the database sequences, their closest relatives are unidentified bacteria. This indicated that most of the rumen bacteria in goats have not been identified. Our results agree previous reports with regard to the presence of bacteria in the rumen of dairy animals, where bacteria were analyzed using a combination of DGGE and sequencing (Kocherginskaya et al., 2001). A positive relationship was observed between the gender-related ruminal metabolism and microbial composition; however SDG supplementation had similar physiological effects on ruminal flora for both genders. The results demonstrate that rumen microorganisms efficiently convert SDG to END and ENL. In turn, SDG and/or the two main mammalian lignans may possess a beneficial effect on rumen fermentation by increasing the VFAs and MCP. Analysis of DGGE also showed that SDG supplement stimulates the growth of R. gnavus, which play a role in the glucuronidase activity in rumen. Thus, SDG has a potential for being used as a feed additive in ruminants. Acknowledgments This work was supported by the National Natural Science Foundation of China (Grant Nos. 30371041 and 30530560). We acknowledge the technical assistance provided by Su Yong and Lü Xuejiao. References Adlercreutz, H., Van der Wildt, J., Kinzel, J., Attalla, H., Wähälä, K., Mäkelä, T., Hase, T., Fotsis, T., 1995. Lignan and isoflavonoid conjugates in human urine. J. Steroid Biochem. Mol. Biol. 52, 97–103. Beaud, D., Tailliez, P., Anba-Mondoloni, J., 2005. Genetic characterization of the ˇ-glucuronidase enzyme from a human intestinal bacterium, Ruminococcus gnavus. Microbiology 151, 2323–2330. Bowey, E., Adlercreutz, H., Rowland, I., 2003. Metabolism of isoflavones and lignans by the gut microflora: a study in germ-free and human flora associated rats. Food Chem. Toxicol. 41, 631–636. Clavel, T., Henderson, G., Alpert, C.A., Philippe, C., Rigottier-Gois, L., Doré, J., Blaut, M., 2005. Intestinal bacterial communities that produce active estrogen-like compounds enterodiol and enterolactone in humans. Appl. Environ. Microbiol. 71, 6077–6085. Clavel, T., Lippman, R., Gavini, F., Doré, J., Blaut, M., 2007. Clostridium sacchargumia sp. Nov. and Lactonifactor longoviformis gen. nov., sp. Nov. two novel human faecal bacteria involved in the conversion of the dietary phytoestrogen secoisolariciresinol diglucoside. Syst. Appl. Microbiol. 30, 16–26. Collier, C.T., Smiricky-Tjardes, M.R., Albin, D.M., Wubben, J.E., Gabert, V.M., Deplancke, B., Bane, D., Anderson, D.B., Gaskins, H.R., 2003. Molecular ecological analysis of porcine ileal microbiota responses to antimicrobial growth promoters. J. Anim. Sci. 81, 3035–3045.

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