Flavonoid-converting capabilities of Clostridium butyricum

Abstract

Clostridium butyricum inhabits various anoxic environments, including soil and the human gut. Here, this common bacterium comes into contact with abundant plant-derived flavonoids. Metabolization of these bioactive polyphenols has been studied in recent years, particularly focusing on gut bacteria due to the proposed health-promoting properties of these dietary constituents. Based on an initial report in 1997 on eriodictyol degradation (Miyake et al. 1997, J Agric Food Chem, 45:3738-3742), the present study systematically investigated C. butyricum for its ability to convert a set of structurally diverse flavonoids. Incubation experiments revealed that C. butyricum deglycosylated flavonoid O-glucosides but only when glucose was absent. Moreover, aglycone members of flavone, flavanone, dihydrochalcone, and flavanonol subclasses were degraded. The C-ring cleavage of the flavanones, naringenin and eriodictyol, was stereospecific and finally resulted in formation of the corresponding hydroxyphenylpropionic acids. Stereospecific C-ring cleavage of the flavanonol taxifolin led to taxifolin dihydrochalcone. C. butyricum did neither cleave flavonols and isoflavones nor catalyze de-rhamnosylation, demethylation, or dehydroxylation of flavonoids. Genes encoding potential flavonoid-metabolizing enzymes were detected in the C. butyricum genome. Overall, these findings indicate that C. butyricum utilizes flavonoids as alternative substrates and, as observed for the dihydrochalcone phloretin, can eliminate growth-inhibiting flavonoids through degradation. KEY POINTS: • Clostridium butyricum deglycosylated flavonoid O-glucosides. • Clostridium butyricum converted members of several flavonoid subclasses. • Potential flavonoid-metabolizing enzymes are encoded in the C. butyricum genome.

Keywords: Clostridium butyricum; Flavonoid; Flavonoid glycoside; Gut bacterium; Polyphenol; Soil bacterium.

Fig. 1

Structures of flavonoids used in the study

Fig. 2

Deglycosylation of flavonoids by C. butyricum was inhibited by glucose present in medium. a, b, and c Incubation of flavonoids with C. butyricum in RCM_mod with glucose: a isoquercetin (quercetin-3-O-glucoside, Q3G), b apigenin-7-O-glucoside (A7G), and c naringenin (NAR). d, e, and f Incubation of flavonoids with C. butyricum in RCM_mod without glucose: d Q3G, e A7G, and f NAR. As controls (Ctrl), flavonoids were incubated in RCM_mod without glucose in the absence of C. butyricum. Data points are means of duplicates. QUE, quercetin; API, apigenin; 4-HPP, 3-(4-hydroxyphenyl)propionic acid

Fig. 3

Conversion of flavonoids by C. butyricum in RCM_mod without glucose: a isoquercetin (quercetin-3-O-glucoside, Q3G) to quercetin (QUE), b directly added QUE was not converted, c taxifolin (TAX) to taxifolin dihydrochalcone (TAX-DHC), d apigenin-7-O-glucoside (A7G) to 3-(4-hydroxyphenyl)propionic acid (4-HPP), e apigenin (API) via naringenin (NAR) to 4-HPP, f NAR to 4-HPP, g phloretin (PHL) to 4-HPP, and h eriodictyol (ERI) to 3-(3,4-dihydroxyphenyl)propionic acid (3,4-DPP). As controls (Ctrl), flavonoids were incubated in medium in the absence of C. butyricum. Data points are means of triplicates; SEM values were < 8%

Fig. 4

The C-ring cleavage of flavanone and flavanonol members by C. butyricum was stereospecific. Chiral HPLC analysis of conversion of racemic a naringenin (NAR), b eriodictyol (ERI), and c taxifolin (TAX) based on individual incubations in RCM_mod without glucose shown in Fig. 3. d Conversion of NAR based on the incubation in RCM_mod with glucose shown in Fig. 2. Percentage values refer to initial concentrations

Fig. 5

Stereopreference in flavanone and flavanonol degradation by C. butyricum differed from that of E. ramulus. Chiral HPLC chromatograms of samples collected in the course of conversion of a naringenin (NAR), b eriodictyol (ERI), and c taxifolin (TAX) by C. butyricum (Cb) or by the flavanone/flavanonol-cleaving reductase (Fcr) from E. ramulus. Samples of conversions by C. butyricum are from experiments conducted with glucose-free RCM_mod

Fig. 6

The growth of C. butyricum was inhibited by phloretin until it was degraded. a Growth of C. butyricum in RCM_mod with glucose in the presence of naringenin (NAR) or phloretin (PHL). Control (Ctrl) incubation with the solvent DMSO only. b Monitoring of PHL conversion to 3-(4-hydroxyphenyl)propionic acid (4-HPP) by C. butyricum in parallel to its growth. Broken line refers to the y axis on the right. Data points are means (± SEM) of triplicates. OD, optical density at 600 nm

Fig. 7

Pathways of flavonoid conversion and involved enzymes in human gut bacteria. Reactions observed in C. butyricum are highlighted with blue arrows. CHI, chalcone isomerase; Fcr, flavanone/flavanonol-cleaving reductase; FLR, flavone reductase; Glu, O-glucosidase; Phy, phloretin hydrolase; Rh, rhamnosidase