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Abstract Eubacterium ramulus, a quercetin-3-glucoside-
degrading anaerobic microorganism that occurs at num-
bers of approximately 108/g dry feces in humans, was tested
for its ability to transform other flavonoids. The organism
degraded luteolin-7-glucoside, rutin, quercetin, kaempferol,
luteolin, eriodictyol, naringenin, taxifolin, and phloretin
to phenolic acids. It hydrolyzed kaempferol-3-sorphoro-
side-7-glucoside to kaempferol-3-sorphoroside and trans-
formed 3,4-dihydroxyphenylacetic acid, a product of anaer-
obic quercetin degradation, very slowly to non-aromatic
fermentation products. Luteolin-5-glucoside, diosmetin-7-
rutinoside, naringenin-7-neohesperidoside, (+)-catechin,
and (–)-epicatechin were not degraded. Cell extracts of
E. ramulus contained α- and β-D-glucosidase activities,
but were devoid of α-L-rhamnosidase activity. Based on
the degradation patterns of these substrates, a pathway for
the degradation of flavonoids by E. ramulus is proposed.
Key words Eubacterium ramulus · Degradation ·
Quercetin · Kaempferol · Luteolin · Eriodictyol ·
Naringenin · Taxifolin · Catechin · Glycosides
Introduction
Flavonoids are widely distributed throughout the plant
kingdom and are taken up in considerable amounts with
food. Beneficial effects for human health have been re-
ported (Wiltrout and Hornung 1988; Frankel et al. 1993).
Although it is known that the intestinal microflora par-
ticipate in the degradation of flavonoids (Nakagawa et al.
1965; Griffiths and Barrow 1972; Baba et al. 1981), there is
a lack of information about the microorganisms and the
mechanisms involved. Schneider et al. (1999) isolated
strains of Eubacterium ramulus from human feces that are
capable of degrading quercetin-3-glucoside to 3,4-dihy-
droxyphenylacetic acid (3,4-DIOHA), butyrate, and acetate.
Phloroglucinol was formed as an intermediate. Simmering
et al. (1999) detected E. ramulus in fecal samples from each
of 20 persons tested at cell numbers of 4.4 × 107–2.0 ×
109/g dry weight. Therefore E. ramulus may be considered
as a key organism for flavonoid degradation in the human
intestinal tract. The aim of this study was to examine the
ability and the structural requirements for the degradation
of flavonoids by E. ramulus. Based on the data obtained,
a mechanism for the degradation of quercetin is proposed.
Materials and methods
Degradation experiments
The degradation experiments were essentially performed as de-
scribed by Schneider et al. (1999), but, instead of quercetin-3-glu-
coside, the compounds listed in Table 1 were added to the defined
bicarbonate-buffered medium B in 16-ml fermentation tubes at con-
centrations of 1 mM or 5 mM. Glucose (10 mM) was added as in-
dicated. The media were inoculated with 1 % of an exponentially
growing culture of E. ramulus (wK1) previously isolated by Schnei-
der et al. (1999). The cultures were incubated under strictly anoxic
conditions with a gas phase of N2 and CO2 (80:20, v/v) at 37°C on
a rotary shaker (170 rpm). After 0, 24, 72 h and when indicated,
1-ml aliquots were taken and immediately stored at –80°C until
they were used for HPLC analyses.
HPLC
Flavonoids and aromatic degradation products were determined as
described by Schneider et al. (1999) using an HPLC with a diode
array detector (Gynkotek, Munich, Germany). Some compounds
were identified by an HPLC-coupled tandem mass spectrometry
(LC-MS/MS). The HPLC (Waters, Milford, USA) used for the lat-
ter method was equipped with a pump (model alliance 2690), an
autosampler (model alliance 2690), and a nucleosil reversed-phase
C18 column (250 × 4.6 mm) with a particle size of 5 µm (Waters).
The HPLC was connected with a tandem quadrupol mass spectrom-
eter, model VG Quattro II, with electrospray ionization (Micro-
Heiko Schneider · Michael Blaut
Anaerobic degradation of flavonoids by Eubacterium ramulus
Arch Microbiol (2000) 173:71–75©Springer-Verlag 2000
Received: 1 July 1999 / Accepted: 25 September 1999
SHORT COMMUNICATION
H. Schneider · M. Blaut
Gastrointestinale Mikrobiologie,
Deutsches Institut für Ernährungsforschung (DIfE)
and Institut für Ernährungswissenschaft der Universität Potsdam,
Arthur-Scheunert-Allee 114–116,
D-14558 Bergholz-Rehbrücke, Germany