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Keywords:

  • anaerobic metabolism;
  • catechin;
  • Clostridium orbiscindens;
  • Eggerthella lenta;
  • epicatechin;
  • flavan-3-ols;
  • Flavonifractor plautii

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To isolate and characterize bacteria from the human intestine that are involved in the conversion of catechins, a class of bioactive polyphenols abundant in the human diet.

Methods and Results:  Two bacterial strains, rK3 and aK2, were isolated from an epicatechin-converting human faecal suspension. The isolates catalysed individual steps in the degradation of (−)-epicatechin and (+)-catechin. Based on their phenotypic characteristics and 16S rRNA gene sequences, the isolates were identified as Eggerthella lenta and Flavonifractor plautii (formerly Clostridium orbiscindens). Eggerthella lenta rK3 reductively cleaved the heterocyclic C-ring of both (−)-epicatechin and (+)-catechin giving rise to 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol. The conversion of catechin proceeded five times faster than that of epicatechin. Higher (epi)catechin concentrations led to an accelerated formation of the ring fission product without affecting the growth of Eg. lenta rK3. Flavonifractor plautii aK2 further converted 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol to 5-(3,4-dihydroxyphenyl)-γ-valerolactone and 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid. Flavonifractor plautii DSM 6740 catalysed the identical reaction indicating it is not strain specific.

Conclusions:  The conversion of dietary catechins by the isolated Eg. lenta and F. plautii strains in the human intestine may affect their bioavailability.

Significance and Impact of the Study:  The majority of catechin metabolites are generated by the intestinal microbiota. The identification of catechin-converting gut bacteria therefore contributes to the elucidation of the bioactivation and the health effects of catechins.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Catechins belong to the flavonoid subclass of flavan-3-ols and are present in a wide range of dietary plants as monomers or as oligomeric and polymeric proanthocyanidins. Main sources of catechins include green and black tea, apples, plums, cocoa and red wine (Arts et al. 2001a; Auger et al. 2004; Gu et al. 2004; Song and Chun 2008). A high intake of catechins was found to be associated with a lower risk of cardiovascular diseases (Arts et al. 2001b,c) and colorectal cancer (Arts et al. 2002; Theodoratou et al. 2007; Simons et al. 2009). Chemopreventive effects of monomeric and oligo/polymeric catechins have been demonstrated in animal experiments (Weyant et al. 2001; Nomoto et al. 2004; Gosse et al. 2005). So far, it has not fully been elucidated whether the observed effects are mediated by the ingested catechins or their metabolites. Antioxidative, anti-inflammatory, antiproliferative and antiplatelet aggregation properties have also been described for catechin metabolites (Koga and Meydani 2001; Unno et al. 2003; Rechner and Kroner 2005; Schroeter et al. 2006; Larrosa et al. 2009). Methylated, glucuronidated and sulfated derivatives of the parent compounds as well as valerolactones and phenolic acids were detected in blood and urine following the oral intake of catechins by rats and humans (Piskula and Terao 1998; Baba et al. 2001a,b; Gonthier et al. 2003; Rios et al. 2003; Tsang et al. 2005; Serra et al. 2010). In vitro studies with human faecal suspensions revealed that the majority of catechin degradation products, including valerolactones and phenolic acids, are generated by the intestinal microbiota (Meselhy et al. 1997; Deprez et al. 2000; Aura et al. 2008; Tzounis et al. 2008; Appeldoorn et al. 2009; Stoupi et al. 2010). Because the absorption of monomeric catechins in the small intestine is not complete and greatly impaired by polymerization, these polyphenols reach the colon and are converted by the resident microbiota (Manach et al. 2005).

While the mechanism of the initial attack of oligomeric and polymeric catechins by gut bacteria remains to be elucidated, the pathway of the conversion of monomers is largely known. The degradation of (epi)catechin starts with fission of the heterocyclic C-ring followed by the formation of hydroxyphenylvalerolactones and hydroxyphenylvaleric acids, which are further converted to hydroxyphenylpropionic acids (Meselhy et al. 1997; Aura et al. 2008; Stoupi et al. 2010; Takagaki and Nanjo 2010). However, knowledge about the gut bacteria involved in the conversion of catechins is so far limited to only one strain isolated from human faeces. Eggerthella lenta SDG-2 catalyses the cleavage of the C-ring of monomeric catechins followed by dehydroxylation of the B-ring (Wang et al. 2001). Human gut bacteria cleaving the C-ring of flavonoids other than catechins, such as flavanonols and flavanones, do not act on (epi)catechin (Winter et al. 1989; Schneider and Blaut 2000; Braune et al. 2001; Schoefer et al. 2003). Herein, we describe two new bacterial isolates from human faeces that catalyse individual steps in the conversion of monomeric catechins.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Chemicals, bacterial strains, media and culture conditions

(−)-Epicatechin and (+)-catechin were purchased from Roth (Karlsruhe, Germany). For preparation of 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (3,4-diHPP-2-ol), a grape seed extract rich in catechins (exGrape SEED OPC40; Breko, Bremen, Germany) was incubated at 10 mg ml−1 with a 1% human faecal suspension in a defined bicarbonate-buffered medium (medium B) (Schneider et al. 1999) supplemented with 100 mmol l−1 glucose. After 48 h of anoxic incubation at 37°C, the culture was centrifuged at 14 000 g for 5 min. Aliquots (200 μl) of the supernatant were applied to the HPLC system, and the fractions containing 3,4-diHPP-2-ol were manually collected, pooled and dried by vacuum centrifugation (RC 10.22.; Jouan, Saint-Nazaire, France).

Eggerthella lenta DSM 2243, Eubacterium plautii DSM 4000 (reassigned Flavonifractor plautii DSM 4000T) and Clostridium orbiscindens DSM 6740 (reassigned Flavonifractor plautii DSM 6740) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ, Braunschweig, Germany).

Brain Heart Infusion (BHI) broth (Roth) supplemented with 0·5 g l−1 cysteine hydrochloride (Merck, Darmstadt, Germany) in 16-ml Hungate tubes sealed with flange-type butyl rubber septum stoppers and open top plastic screw caps (Bellco Glass, Vineland, NJ, USA) and gassed with N2/CO2 (80/20%, v/v) was used for the isolation, strain maintenance and degradation experiments. Bacterial growth was monitored by measuring the optical density at 600 nm (OD600). Wilkins Chalgren Anaerobe (WCA) agar (Oxoid, Basingstoke, UK) was used for plating of mixed cultures and isolating single colonies. Pure cultures were grown on Columbia agar with 5% sheep blood (bioMérieux, Marcy l’Etoile, France). Preparation of faecal suspensions and incubation of agar plates were performed under strictly anoxic conditions in an anoxic workstation (MACS Anaerobic Workstation; Don Whitley Scientific Ltd., Shipley, UK) containing a gas atmosphere of N2/CO2/H2 (80/10/10%, v/v/v).

Isolation procedure

A fresh faecal sample from a healthy human donor who had not taken antibiotics in the 6 months prior to collection was immediately processed under anoxic conditions. One gram of faeces was resuspended in 9 ml BHI broth followed by centrifugation at 300 g for 1 min. An aliquot of 500 μl of the resulting supernatant was added to 4·5 ml of BHI broth supplemented with 2 mmol l−1 epicatechin and incubated at 37°C in a tube rotator at 20 rev min−1 (Stuart SB2; Bibby Scientific, Staffordshire, UK). After 24 h, fresh 10 ml BHI broth supplemented with 2 mmol l−1 epicatechin were inoculated with 100 μl of the culture. After 24 h of growth, the culture was serially diluted (10−1 to 10−8) in BHI broth supplemented with epicatechin (1 mmol l−1) and tetracycline (10 μg ml−1; Roth). Tetracycline was added to support the enrichment of epicatechin-converting bacteria by inhibiting the growth of other bacterial community members. We had previously observed that among several antibiotics tested at varying concentrations only tetracycline at concentrations of up to 10 μg ml−1 hardly impaired the formation of metabolites from epicatechin. Increased tetracycline concentrations (15, 20, 25, 40 μg ml−1) had resulted in a largely delayed conversion of epicatechin by bacterial suspensions. In spite of these preliminary tests, a loss of active tetracycline-sensitive bacteria could not be excluded using this approach. The resulting cultures were incubated at 37°C for 5 days. From the highest dilution showing epicatechin conversion (10−4), serial dilutions (10−1 to 10−5) were prepared and incubated as described before. Aliquots of the most diluted active culture were plated on WCA agar and incubated at 37°C for 48 h. Colonies were tested in BHI broth for their ability to convert epicatechin. Pure cultures were subsequently obtained by repeated streaking on WCA agar.

Phenotypic characterization

Gram staining and the KOH string test were performed as described elsewhere (Reddy 2007). The KOH string test is based on the lysis of Gram-negative cells by KOH and the subsequent release of DNA forming a string. The occurrence of endospores was tested by the Schaeffer-Fulton stain (Reddy 2007) following growth and storage in BHI broth. In addition, BHI agar supplemented with MnSO4·H2O (10 mg l−1), CaCl2·2H2O (100 mg l−1) and MgSO4·7 H2O (500 mg l−1) was used to promote endospore formation. Catalase, oxidase and tryptophanase were tested with Bactident kits (Merck). Further biochemical characteristics were determined using the Vitek system (ANI card) (bioMérieux) according to the manufacturer’s instructions.

16S rRNA gene sequencing

Genomic DNA was extracted from overnight grown cultures with the FastDNA Spin kit (MP Biomedicals, Heidelberg, Germany) following the manufacturer’s instructions. The 16S rRNA genes were almost completely amplified by PCR with the primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1401R (5′-CGGTGTGTACAAGACCC-3′) or 1492R (5′-TACCTTGTTACGACTT-3′) (Nübel et al. 1996; Kageyama et al. 1999) as described previously (Clavel et al. 2005). Following purification with the innuPREP PCRpure Kit (Analytik Jena AG, Jena, Germany), the PCR products were sequenced with the primers used for PCR (Eurofins MWG Operon, Martinsried, Germany). Nucleotide sequences were edited manually with the ContigExpress tool of the VectorNTI Suite 9 software (Invitrogen, Carlsbad, CA, USA) and aligned with similar bacterial 16S rRNA gene sequences obtained with the Blast function of the National Center for Biotechnology Information (NCBI) server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) using the AlignX tool of the VectorNTI Suite 9 software. The 16S rRNA gene sequences of the isolates were submitted to the GenBank database under accession numbers HQ455039 (strain rK3) and HQ455040 (strain aK2).

Degradation experiments

Stock solutions (10 mmol l−1) of (−)-epicatechin and (+)-catechin in dimethyl sulfoxide (DMSO) were sterile filtered and added to 10 ml of BHI broth at a final concentration of 100 μmol l−1. The isolated 3,4-diHPP-2-ol was dissolved in water, quantified using epicatechin as a reference and added to 2 ml of BHI broth at a final concentration of 100 μmol l−1. Overnight grown bacterial cultures were added to 2% (v/v) final concentration. For controls, each substrate and the bacterial strains were incubated separately under the same conditions. The tubes were incubated in a tube rotator (20 rev min−1) at 37°C, and samples were taken at different time points. Aliquots (50 μl) were acidified with 3·5 μl 7·5% HCl, centrifuged at 14 000 g for 5 min and 40 μl of the supernatant was applied to HPLC analysis. For the determination of concentration-dependent effects, catechin and epicatechin were additionally applied at final concentrations of 1·0, 2·5 and 5·0 mmol l−1 using 100-fold concentrated stock solutions in DMSO.

HPLC analysis

HPLC analysis was performed using a Summit HPLC system (Dionex, Idstein, Germany) consisting of a pump (P680A LPG), an autosampler (ASI-100T), a column oven (TCC-100) and a diode array detector (UVD 340U PDA) equipped with a Luna Phenyl-Hexyl column (5 μm, 250 × 4·6 mm) and a Phenyl Security Guard Cartridge (4 × 3·0 mm) (Phenomenex, Aschaffenburg, Germany). The column temperature was maintained at 37°C. Aqueous 2% (v/v) acetic acid (solvent A) and methanol (solvent B) served as the mobile phase in a gradient mode (B from 5 to 40% in 25 min, held for 3 min, from 40 to 100% in 10 min) at a flow rate of 1 ml min−1. Detection was at 280 nm; UV spectra were recorded in the range of 200–400 nm. For quantification, calibration curves of epicatechin and catechin were used. Concentrations of 3,4-diHPP-2-ol were estimated based on the calibration curve of epicatechin, because a reference compound is not available. For control of the HPLC system and data processing, the Chromeleon 6.40 software (Dionex, Sunnyvale, CA, USA) was used.

UPLC-ESI-MS analysis

Metabolites were further characterized by ultra performance liquid chromatography (UPLC)-coupled mass spectrometry (MS) using either complete supernatants from degradation experiments or metabolite-containing fractions manually collected after separation by HPLC. The Acquity UPLC system (Waters, Milford, MA) consisted of a solvent and a sample manager, a diode array detector, and a UPLC BEH C18 column (1·7 μm, 50 × 2·1 mm) and was connected to a triple quadrupole mass spectrometer with a Z-spray API electrospray ionization (ESI) source (Quattro Premier XE; Waters). The column temperature was maintained at 27°C. Aqueous 1% (v/v) formic acid (solvent A) and methanol (solvent B) served as the mobile phase in a gradient mode (B from 5 to 40% in 3·10 min, held for 0·40 min, from 40 to 100% in 1·50 min) at a flow rate of 0·35 ml min−1. A 5-μl aliquot of the sample was injected. MS analyses were carried out in negative and positive ionization mode using a capillary voltage of 1·8 and 0·7 kV, respectively, a source block temperature of 100°C and a desolvation temperature of 450°C. The cone voltage was adjusted to 25 V. MS/MS analyses were performed in negative ionization mode using argon at a pressure of 3·1 × 10−1 Pa for collision-induced dissociation (CID). The compound-dependent collision energy was between 10 and 25 eV. Spectra generally covered a mass range from m/z 50 to 500, which was extended to m/z 1050 for analysis of complex samples. Data were analysed using MassLynx 4.1 software (Waters).

Statistical analysis

Means and standard deviations were calculated using Microsoft Excel 2002 (Microsoft Corporation, Redmond, WA, USA). Differences were checked for significance using an unpaired Student’s t-test. P-values <0·05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation and characterization of catechin-converting bacteria

The source of isolates was a faecal suspension from one healthy human donor, which converted epicatechin via the unknown metabolites M1, M2 and M3. While M1 was observed as the first metabolite in the course of epicatechin conversion, M2 and M3 appeared at later time points. The faecal suspension was subjected to serial dilution and enrichment cycles in the presence of epicatechin and tetracycline. Subsequently, a culture was obtained that converted epicatechin to M2 and M3. Gram staining revealed two bacterial morphotypes of Gram-positive and Gram-negative rods. Further separation steps led to the isolation of two pure cultures of strictly anaerobic bacteria, strain rK3 and strain aK2. Strain rK3 was a coccoid Gram-positive rod occurring singly or in pairs. The straight rods of strain aK2 appeared Gram negative based on both staining and the KOH string test. After 3 days of growth on sheep blood agar at 37°C, both isolates formed nonhaemolytic, circular, entire, raised, opaque, shiny colonies that were <1 mm in diameter. Endospores were observed neither in complex media nor on agar supplemented with adequate levels of divalent cations to promote spore formation. The isolates were devoid of catalase, oxidase and tryptophanase. Strain rK3 was positive for arginine dihydrolase and hydrolysis of l-lysine-p-nitroanilide. Strain aK2 hydrolysed p-nitrophenyl phosphate and p-nitroanilide derivatives of l-leucine, l-alanine and l-lysine.

Identification of the catechin-converting bacterial isolates

The 16S rRNA gene sequence of both isolates was determined for the most part and compared with corresponding bacterial sequences in databases. The obtained 16S rRNA gene sequence of strain rK3 (1282 bp, accession number HQ455039) was almost completely identical to that of several strains of Eg. lenta, such as Eg. lenta SECO-Mt75m2 (AY937380, 99·8% identity), Eg. lenta SDG-2 (EF413638, 98·7%) and Eg. lenta DSM 2243T (AF292375, 98·5%). Other Eggerthella species revealed considerably lower identity values of their 16S rRNA gene sequence in relation to that of strain rK3 including Eggerthella sinensis DSM 16107T (AY321958, 95·4%). Based on its phylogenetic position and its phenotypic characteristics, which matched those of the type strain of Eg. lenta (DSM 2243), strain rK3 belongs to the species Eg. lenta.

The alignment of the 16S rRNA gene sequence of strain aK2 (1420 bp, accession number HQ455040) revealed 99·9% identity to Cl. orbiscindens DSM 6740T (Y18187), 99·6% identity to Eu. plautii CCUG 28093T (=DSM 4000T, AY724678) and 97·0% identity to Bacteroides capillosus ATCC 29799 (AY136666). The phenotypic characteristics determined in parallel for strain aK2 and the type strains of Cl. orbiscindens (DSM 6740) and Eu. plautii (DSM 4000) were largely consistent including their inability to form spores. However, differences were observed in Gram behaviour and an enzymatic activity. While strain aK2 and Cl. orbiscindens stained Gram negative, Eu. plautii was Gram positive according to staining. Based on the KOH string test, strain aK2 and Eu. plautii were Gram negative, whereas Cl. orbiscindens appeared Gram positive. Furthermore, l-alanine-p-nitroanilide was hydrolysed by both strain aK2 and Cl. orbiscindens, but not by Eu. plautii. Based on these phylogenetic and phenotypic analyses, the newly isolated strain aK2 displayed the highest similarity to Cl. orbiscindens. However, Cl. orbiscindens and Eu. plautii have recently been unified in a new genus as F. plautii with the type strain DSM 4000T, whereas the related B. capillosus has been reassigned to Pseudoflavonifractor capillosus (Carlier et al. 2010). Thus, strain aK2 belongs to the species F. plautii.

Catechin conversion by the bacterial isolates

Following their isolation from the epicatechin-converting mixed culture, Eg. lenta rK3 and F. plautii aK2 were tested in pure culture for their ability to transform (−)-epicatechin and (+)-catechin. While the compounds were stable during their incubation in the absence of bacteria, both catechins were completely converted by Eg. lenta rK3. However, the conversion rates of epicatechin and catechin differed considerably (Fig. 1a,b). After 8 h of incubation, catechin was almost completely used up, whereas 40 μmol l−1 of the initially applied 100 μmol l−1 epicatechin was still available. The reduced recovery of the catechins at the beginning of the incubation irrespective of the presence of bacteria may have been caused by binding of these compounds to constituents of the culture medium. This phenomenon was already described previously (Hofmann et al. 2006). The conversion of both epicatechin and catechin resulted in the formation of metabolite M1 subsequently characterized as 1-(3,4-dihydroxyphenyl)-3-(2,4,6-trihydroxyphenyl)propan-2-ol (3,4-diHPP-2-ol, structure in Fig. 3). In accordance with the initial conversion of the catechins, 3,4-diHPP-2-ol was formed more rapidly from catechin than from epicatechin (Fig. 1a,b). The initial rate of 3,4-diHPP-2-ol formation increased with rising concentration of epicatechin or catechin in the range of 0·1–5·0 mmol l−1 (Table 1), reaching significance only for catechin (P < 0·01). At the various concentrations, the formation of 3,4-diHPP-2-ol from catechin proceeded 4·1–5·4 times faster than that from epicatechin (P < 0·0005). Up to the maximal concentration of 5 mmol l−1, neither catechin nor epicatechin affected the growth of Eg. lenta rK3 (Table 1). The incubation of 3,4-diHPP-2-ol added directly to Eg. lenta rK3, did not result in its conversion.

image

Figure 1.  Conversion by Eggerthella lenta rK3 of (+)-catechin (•) (a) and (−)-epicatechin (•) (b) to 3,4-diHPP-2-ol (M1, bsl00066). (+)-Catechin (○) (a) and (−)-epicatechin (○) (b) without bacteria served as controls. Values are means ± SD.

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image

Figure 3.  Proposed conversion of (+)-catechin and (−)-epicatechin by the isolated human intestinal bacteria.

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Table 1.   Initial rates of 3,4-diHPP-2-ol formation, maximal 3,4-diHPP-2-ol concentrations and cell densities (OD600) in the course of incubation of Eggerthella lenta rK3 with varying concentrations of (−)-epicatechin (EC) or (+)-catechin (CA)
Substrate concentration (mmol l−1)Initial 3,4-diHPP-2-ol formation (μmol l−1 h−1)*Maximal 3,4-diHPP-2-ol concentration (μmol l−1)†OD600 at 4 hOD600 at 8 h
ECCAECCAECCAECCA
  1. Values are means ± SD, n = 3.

  2. NA, not applicable.

  3. *During the first 4 h of incubation.

  4. †Within 100 h (EC) or 74 h (CA) of incubation: maximal values for 0·1 mmol l−1 EC at 24 h; for 1·0, 2·5 and 5·0 mmol l−1 EC at 100 h; for 0·1 mmol l−1 CA at 8 h; for 1·0, 2·5 and 5·0 mmol l−1 EC at 74 h.

  5. ‡Different from the respective EC value (< 0·0005).

0NANANANA0·01 ± 0·020·01 ± 0·010·06 ± 0·010·10 ± 0·01
0·11·9 ± 0·710·3 ± 0·6‡37·7 ± 3·543·2 ± 1·00·02 ± 0·020·02 ± 0·010·06 ± 0·010·11 ± 0·01
1·02·5 ± 0·213·3 ± 0·5‡143·3 ± 15·1178·9 ± 5·10·04 ± 0·020·03 ± 0·010·07 ± 0·020·10 ± 0·01
2·53·0 ± 0·414·4 ± 0·3‡224·5 ± 3·3222·0 ± 12·10·00 ± 0·010·04 ± 0·030·06 ± 0·020·09 ± 0·02
5·03·6 ± 1·014·6 ± 1·0‡192·3 ± 9·7250·4 ± 12·10·01 ± 0·010·03 ± 0·020·08 ± 0·010·10 ± 0·01

Flavonifractor plautii aK2 converted neither catechin nor epicatechin within 70 h of incubation. Thus, the isolate was tested for its ability to metabolize 3,4-diHPP-2-ol, the product of (epi)catechin conversion by Eg. lenta rK3. Flavonifractor plautii aK2 completely converted 3,4-diHPP-2-ol (100 μmol l−1) within 6 h of incubation, whereas the compound was stable in the absence of bacteria (Fig. 2a). Two metabolites, M2 and M3, were first detected after 4 and 6 h, respectively, of incubation with F. plautii aK2. The concentration of M2 was maximal after 6 h and slightly decreased afterwards. The concentration of M3 increased rapidly until 8 h followed by a minor increase during further incubation. Characterization of the observed metabolites by MS analyses and taking literature data into account led to the assignment of M2 as 5-(3,4-dihydroxyphenyl)-γ-valerolactone (3,4-diHPVL) and M3 as 4-hydroxy-5-(3,4-dihydroxyphenyl)valeric acid (4-H-3,4-diHPVA) (structures in Fig. 3).

image

Figure 2.  Conversion of 3,4-diHPP-2-ol by Flavonifractor plautii aK2 (a) and F. plautii DSM 6740 (b). Both strains transformed 3,4-diHPP-2-ol (•) to 3,4-diHPVL (M2, ◆) and 4-H-3,4-diHPVA (M3, bsl00066). 3,4-diHPP-2-ol without bacteria (○) served as a control. Broken lines refer to the y-axis on the right. Values are means ± SD.

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To elucidate whether the cleavage of 3,4-diHPP-2-ol is a specific feature of the newly isolated strain aK2, F. plautii DSM 6740 (formerly Cl. orbiscindens DSM 6740T) was tested for its ability to convert 3,4-diHPP-2-ol. Flavonifractor plautii DSM 6740 completely transformed 3,4-diHPP-2-ol within 4 h of incubation to 3,4-diHPVL and 4-H-3,4-diHPVA (Fig. 2b). The metabolite concentrations were similar to those observed for F. plautii aK2 (Fig. 2a). In contrast to the isolate, F. plautii DSM 6740 formed 3,4-diHPVL and 4-H-3,4-diHPVA nearly simultaneously from 3,4-diHPP-2-ol (Fig. 2b).

Characterization and structural assignment of bacterial (epi)catechin metabolites

In HPLC-DAD analysis, M1 (Rt = 17·0 min) eluted closely to epicatechin (Rt = 17·8 min, cf. Rt = 12·8 min for catechin) and exhibited a UV spectrum virtually identical to those of epicatechin and catechin (λmax = 279 nm). M2 and M3 had identical absorption maxima at 281 nm, but different retention times of 19·8 and 13·0 min, respectively. The UV spectra of M2 and M3 were similar but not identical to each other and to (epi)catechin.

ESI-MS analysis of M1-containing fractions in the negative mode gave a signal for the deprotonated molecule at m/z 291 [M–H]. The product-ion spectrum for m/z 291 using a collision energy of 25 eV revealed a base peak at m/z 123 and additional fragments at m/z 135 (36% base peak intensity, BPI), 167 (31%), 151 (19%) and 109 (15%). Further characteristic CID fragments described previously for this compound (Stoupi et al. 2010) were found at lower levels, such as m/z 205 (8%) and m/z 247 (5%). The molecular mass of 292 for M1 was confirmed by positive ESI-MS analysis showing the protonated molecule at m/z 293 and the analogous [M+Na]+ cluster at m/z 315. The mass difference of 2 u compared with the deprotonated molecule at m/z 289 [M–H] and the protonated molecule at m/z 291 [M+H]+ of (epi)catechin indicated that M1 was formed from these compounds by reduction. Based on this data and reports in the literature (Meselhy et al. 1997; Wang et al. 2001; Appeldoorn et al. 2009; Stoupi et al. 2010), we conclude M1 to be 3,4-diHPP-2-ol.

The metabolites M2 and M3 were analysed in nonfractionated degradation supernatants. MS analysis of M2 revealed a deprotonated molecule at m/z 207 [M–H]. In the positive mode, the corresponding protonated molecule at m/z 209 [M+H]+ and the [M+Na]+ cluster at m/z 231 were observed. Using a collision energy of 15 eV, further CID fragmentation of the deprotonated molecule m/z 207 resulted in two main peaks at m/z 163 (base peak) and 122 (31% BPI). The most intense fragment m/z 163 [M–H–CO2] has been described previously for a metabolite of bacterial catechin conversion (Meng et al. 2002; Appeldoorn et al. 2009; Stoupi et al. 2010), which was subsequently identified by NMR analysis as 3,4-diHPVL (Meselhy et al. 1997; Li et al. 2000; Kohri et al. 2003; Unno et al. 2003). In addition, the obtained minor fragments at m/z 108 (6% BPI) and 85 (5%) presumably represent fragments of the B-ring and the lactone ring, respectively, and indicate that M2 is 3,4-diHPVL.

For M3, the deprotonated molecule at m/z 225 [M–H] was observed by negative ionization. The corresponding molecular mass of 226 is in agreement with 4-H-3,4-diHPVA, an epicatechin metabolite described in previous studies following MS and NMR analyses (Kohri et al. 2003; Stoupi et al. 2010). MS/MS analysis of m/z 225 [M–H] revealed fragment ions at m/z 101 (base peak), 123 (87% BPI), 163 (60%), 207 (13%) and 57 (7%). The observed fragmentation differed from that reported previously for a compound tentatively identified as 4-H-3,4-diHPVA (Stoupi et al. 2010). The base peak m/z 101 observed under the present conditions using a collision energy of 15 eV may represent a deprotonated side chain fragment [C4H5O3], which can be decarboxylated further as indicated by the signal at m/z 57. The daughter ion at m/z 123 corresponds to the dihydroxybenzyl fragment, while the peaks at m/z 207 and 163 presumably resulted from the loss of water [M–H–H2O] followed by loss of CO2. The full scan of M3 in positive mode included the expected protonated molecule m/z 227 [M+H]+ at very low intensity, whereas major peaks appeared at m/z 249 and 209. While the former is the suspected Na adduct [M+Na]+, the latter fragment may have resulted from the loss of water. Based on our analyses and on literature data (Kohri et al. 2003; Llorach et al. 2009), M3 is tentatively identified as 4-H-3,4-diHPVA.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In the present study, two bacterial strains were isolated from human faeces that catalyse different steps in (epi)catechin conversion. The isolates were identified as strains of Eg. lenta and F. plautii, respectively. The species F. plautii was created recently by unifying Cl. orbiscindens and Eu. plautii owing to their highly similar 16S rRNA gene sequence (Carlier et al. 2010). Although the two latter species were reported to differ in their ability to form spores, we never observed spores, neither for the isolated F. plautii aK2 nor for the former type strains Cl. orbiscindens DSM 6740 and F. plautii DSM 4000. Eggerthella lenta and Cl. orbiscindens were first isolated from human faeces (Eggerth 1935; Winter et al. 1991), and both are common members of the human intestinal microbiota. Clostridium orbiscindens and Eg. lenta were detected in the faeces of 80% and up to 47%, respectively, of human subjects. The numbers ranged from 108 to 109 cells per gram of faecal dry weight (Finegold et al. 1983; Schwiertz et al. 2000; Schoefer et al. 2003). While the isolated strain Eg. lenta rK3 cleaved the heterocyclic C-ring of both (−)-epicatechin and (+)-catechin to 3,4-diHPP-2-ol, F. plautii aK2 catalysed conversion of the latter compound to 3,4-diHPVL and 4-H-3,4-diHPVA (Fig. 3). Interestingly, plasma nutrikinetic parameters of the C-3 methoxylated metabolite of 3,4-diHPVL following tea consumption by humans were reported to correlate with members of the Actinobacteria and the Clostridium clusters (van Duynhoven et al. 2011). These bacterial groups also comprise Eg. lenta and F. plautii, respectively. The metabolites formed by Eg. lenta rK3 and F. plautii aK2 have already been described earlier for the degradation of monomeric catechins and procyanidins in animal experiments and/or in vitro studies with human faecal microbiota (Meselhy et al. 1997; Kohri et al. 2003; Unno et al. 2003; Tzounis et al. 2008; Appeldoorn et al. 2009; Stoupi et al. 2010). Only 3,4-diHPVL has been detected in human studies following tea ingestion (Li et al. 2000; Lee et al. 2002; Meng et al. 2002; Sang et al. 2008; Del Rio et al. 2010; Roowi et al. 2010). However, information about the bacterial species involved in the conversion of catechins in the gut is largely missing. Only Eubacterium sp. SDG-2 had been demonstrated to catalyse the C-ring cleavage of all (epi)catechin stereoisomers and other derivatives (Wang et al. 2001). This strain was subsequently identified as Eg. lenta (Jin et al. 2007). This human faecal isolate catalyses in addition the dehydroxylation of the ring fission products at C-4 of the B-ring (Wang et al. 2001). Such an activity was not observed for the new isolate Eg. lenta rK3. However, the comparison of the strains’ 16S rRNA gene sequences revealed an identity of nearly 99%.

Eggerthella lenta rK3 converted (+)-catechin much more rapidly than (−)-epicatechin (Fig. 1), as observed previously for human faecal suspensions (Aura et al. 2008; Tzounis et al. 2008). The initial rates of 3,4-diHPP-2-ol formation increased with rising concentration of both (+)-catechin and (−)-epicatechin (Table 1). Whether the enhanced conversion in each case is because of an accelerated enzymatic turnover, an improved transport or both, has to be clarified in future studies. Up to a concentration of 5 mmol l−1 the growth of Eg. lenta rK3 was affected by neither catechin nor epicatechin. As demonstrated previously, the incubation of human faecal microbiota with 0·5 mmol l−1 catechin influenced the growth of certain bacterial groups positively or negatively (Tzounis et al. 2008). However, Eg. lenta was not covered by the probes used in this study for detection of bacteria. In humans, black tea consumption did not significantly affect the profile of bacteria analysed including the Atopobium group, which encompasses Eg. lenta (Mai et al. 2004).

Clostridium orbiscindens is known to degrade flavonols and flavones, which includes the C-ring fission of the intermediary flavanonols and flavanones, respectively (Winter et al. 1989; Schoefer et al. 2003). In contrast, Cl. orbiscindens does not cleave catechins (Winter et al. 1989), which also applies to the new isolate. However, F. plautii aK2 converted the final product of (epi)catechin cleavage by Eg. lenta, 3,4-diHPP-2-ol. The conversion of 3,4-diHPP-2-ol was catalysed not only by F. plautii aK2 but also by F. plautii DSM 6740 (Fig. 2), the former type strain of Cl. orbiscindens. This indicates that the observed activity is not specific to a single strain. The two metabolites, 3,4-diHPVL and 4-H-3,4-diHPVA, were formed with a similar kinetic. However, the former was detected earlier indicating its partial hydrolysis to the hydroxyvaleric acid. The mechanism of the conversion of 3,4-diHPP-2-ol deserves further investigation. In several studies, 3,4-diHPVL was identified as one of the main products of intestinal catechin metabolism, whereas 4-H-3,4-diHPVA was detected less frequently (Li et al. 2000; Kohri et al. 2003; Unno et al. 2003). However, 3,4-diHPVL may be formed also spontaneously from 4-H-3,4-diHPVA at low pH during processing of samples (Takagaki and Nanjo 2010).

The main end products of catechin metabolism of human intestinal microbiota are phenylpropionic acids (Meselhy et al. 1997; Deprez et al. 2000; Aura et al. 2008; Tzounis et al. 2008; Appeldoorn et al. 2009; Stoupi et al. 2010). These phenolic acids are proposed to result from further degradation of 3,4-diHPVL and 4-H-3,4-diHPVA. However, the bacterial species catalysing this conversion have yet to be identified. By degrading the ingested compounds and forming of metabolites, gut bacteria may considerably affect the proposed health effects of dietary catechins. However, knowledge on the biological activity of the microbial metabolites is still limited. Compared with its precursor (−)-epicatechin, 3,4-diHPVL shows a lower antioxidant potential (Unno et al. 2003). The breakdown product 3-(3,4-dihydroxyphenyl)propionic acid has anti-inflammatory effects (Larrosa et al. 2009). The evaluation of the impact of gut bacteria on catechin conversion becomes even more challenging, because catechins may influence the composition of human intestinal microbiota (Tzounis et al. 2008).

In conclusion, the identification of gut bacteria involved in catechin conversion set the basis to unravel the mechanisms underlying the observed enzymatic activities and to correlate the presence of specific bacteria with the metabolism of these bioactive compounds in the intestine. Our data indicate that individual species catalyse only single steps in the degradation pathway, which implies an even more complex situation considering the conversion of complex catechins, such as procyanidins in the gut.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by the Federal Ministry of Education and Research (BMBF, Germany) (grant number 0313828D). We thank Anke Gühler and Sabine Schmidt for technical assistance. Wolfgang Lörsch (Breko) kindly provided the grape seed extract.

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  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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