The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota

International Journal of Food Microbiology 140 (2010) 175–182

Contents lists available at ScienceDirect

International Journal of Food Microbiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j f o o d m i c r o

The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota Dobroslawa Bialonska a,d,⁎, Priya Ramnani c, Sashi G. Kasimsetty a, Kesava R. Muntha a, Glenn R. Gibson c, Daneel Ferreira a,b a

Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, 38677 University, USA National Center for Natural Products Research, Research Institute of Pharmaceutical Sciences, The University of Mississippi, 38677 University, USA Food and Microbial Sciences Unit, School of Food Biosciences, University of Reading, Reading, RG6 6AP, UK d Institute of Environmental Sciences, Jagiellonian University, Gronostajowa 7, 30-387 Krakow, Poland b c

a r t i c l e

i n f o

Article history: Received 28 September 2009 Received in revised form 8 February 2010 Accepted 26 March 2010 Keywords: Punicalagins Pomegranate by-product (POMx) Human gut microbiota Urolithins Short chain fatty acids

a b s t r a c t We have examined the gut bacterial metabolism of pomegranate by-product (POMx) and major pomegranate polyphenols, punicalagins, using pH-controlled, stirred, batch culture fermentation systems reflective of the distal region of the human large intestine. Incubation of POMx or punicalagins with faecal bacteria resulted in formation of the dibenzopyranone-type urolithins. The time course profile confirmed the tetrahydroxylated urolithin D as the first product of microbial transformation, followed by compounds with decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A. POMx exposure enhanced the growth of total bacteria, Bifidobacterium spp. and Lactobacillus spp., without influencing the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group. In addition, POMx increased concentrations of short chain fatty acids (SCFA) viz. acetate, propionate and butyrate in the fermentation medium. Punicalagins did not affect the growth of bacteria or production of SCFA. The results suggest that POMx oligomers, composed of gallic acid, ellagic acid and glucose units, may account for the enhanced growth of probiotic bacteria. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Pomegranate (Punica granatum L.) is grown commercially in the Near East, India, Spain, Israel and the United States (California) where it is of significant economic importance (Seeram et al., 2005). Pomegranate fruits and products, including juice, tea, wine and extracts are widely consumed and recognized for their health benefits. For instance, commercially manufactured pomegranate juice has a higher antioxidant activity than red wine and green tea (Gil et al., 2000). In addition, pomegranate extract inhibits the proliferation of human cancer cells and modulates inflammatory subcellular signal pathways and apoptosis (Seeram et al., 2005). In recent years, most health advantages of pomegranate have been attributed to the presence of ellagitannins, mainly punicalagins1 (Fig. 1) and ellagic acid (Adams et al., 2006; Gil et al., 2000; Reddy et al., 2007; Seeram et al., 2005). During the industrial hydrostatic processing of the whole fruits, ellagitannins are extracted in significant amounts, subsequently enriching pomegranate juice with at least 2 g/L of punicalagins (Seeram et al., 2005). After the first ⁎ Corresponding author. Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, 38677 University, USA. 1 We prefer to use the plural punicalagins, since these compounds exist in solution as the α- and β-anomers as well as the acyclic hydroxyaldehyde analogue. 0168-1605/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2010.03.038

squeezing of pomegranate fruits leading to juice production, the residual material is additionally pressed and extracted with water to afford by-product (POMx) that is commercially available as a dietary supplement. POMx is rich in oligomers composed of 2–10 repeating units of gallic acid, ellagic acid, and glucose, in different combinations. Although pomegranate ellagitannins showed high bioactivity during in vitro assays, they were not detected in human systemic circulation after consumption of large amounts of pomegranate products (Mertens-Talcott et al., 2006; Seeram et al., 2006), and only trace amounts of ellagic acid have been found in human blood (Seeram et al., 2008). At the pH levels of the small intestine and under physiological conditions of human cell lines, punicalagins undergo partial hydrolysis and spontaneous internal lactone formation to yield ellagic acid (Larrosa et al., 2006a). The remaining ellagitannins and ellagic acid are retained unabsorbed in the gut lumen where they likely interact with complex intestinal bacteria. The composition of human gut microbiota has been linked to both health improvement and the development of various diseases. Beneficial bacteria, known as probiotics (e.g., Bifidobacterium, Lactobacillus) provide the following benefits: function as a ‘barrier’ against pathogens; stimulate the host immune system; prevent food allergies and tumours; produce vitamins; metabolize cholesterol and other lipids; and enhance mineral bioavailability (Gibson, 2008; Hord, 2008). Conversely, the overgrowth of deleterious bacterial species

176

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

The aim of the present study was to investigate the potential of pomegranate by-product and punicalagins to influence the growth of specific bacterial groups and short chain fatty acids formation, in a pHcontrolled, stirred, batch-culture fermentation system that is reflective of the environmental conditions located in the distal region of the human large intestines. We also investigate the bacterial transformation of ellagitannins to urolithins. 2. Materials and methods 2.1. Materials

Fig. 1. Chemical structure of punicalagins and ellagic acid.

(e.g., certain members of Clostridium group) causes chronic and acute bowel diseases and has been associated with aging, cancer, obesity and Alzheimer's disease (Onoue et al., 1997; Rastall et al., 2005). Dietary substrates influence gut microbiota by either enhancing the growth of beneficial bacteria or causing their depletion. Phenolic components of common foods readily contribute to gut bacteria modulation (Ahn et al., 1998; Lee et al., 2006; Parkar et al., 2008; Tzounis et al., 2008). In addition, human intestinal bacteria are able to metabolize dietary polyphenols (Lee et al., 2006). Punicalaganis and ellagic acid are transformed by human gut bacteria to the dibenzopyranone-type urolithins (Fig. 2) (Cerda et al., 2004; Espin et al., 2007; Ito et al., 2008; Larrosa et al., 2006b). Despite increased interest by researchers, industry and consumers towards the beneficial health properties of pomegranate, the impact of pomegranate products on human gut microbiota and their potential prebiotic activity has not been recognized. Our previous investigation using pure bacterial isolates showed that pomegranate by-product and punicalagins significantly inhibited the growth of pathogenic Escherichia coli, Pseudomonas aeruginosa (Reddy et al., 2007), as well as clostridia and Staphyloccocus aureus (Bialonska et al., 2009a). Probiotic lactobacilli and most bifidobacteria were generally not affected, while the growth of Bifidobacterium breve and Bifidobacterium infantis was significantly enhanced under POMx treatment (Bialonska et al., 2009a). Owing to the complexity of interactions between ellagitannins and microbiota in the intestinal environment, determined by the abundance and type of bacterial strains, the results obtained using pure isolates should be verified in a more relevant system.

The commercial extract of pomegranate by-product (POMx) (100 mL) was provided by POM Wonderful, Los Angeles, California, in January 2008. Unless otherwise stated, all chemicals and reagents were obtained from Sigma-Aldrich Co. Ltd (Poole, Dorset, UK or St. Louis, MO, USA) or Fisher Scientific (Loughborough, Leics, UK or Pittsburgh, PA, USA). All the nucleotide probes used for fluorescent in situ hybridisation (FISH) were commercially synthesised and labelled with the fluorescent dye Cy3 at the 5′ end (Sigma Aldrich Co. Ltd., UK). Sterilisation of media and instruments was done by autoclaving at 121 °C for 15 min. 2.2. Isolation and characterization of punicalagins Extraction of punicalagins was performed by the procedure described previously by use of a step gradient consisting of increasing amounts of methanol in water (Reddy et al., 2007). POMx (100 mL) was diluted to 500 mL with Millipore purified water and successively partitioned with ethyl acetate (3 × 200 mL) and n-butanol (3 × 200 mL). The n-butanol extract (2.0 g) was concentrated and subjected to Amberlite XAD-16 column chromatography (500 g, 6 × 35 cm) and eluted with water (2.0 L) and methanol (2.0 L), successively. The methanol fraction on removal of solvent under reduced pressure afforded a tannin fraction (1.3 g). This was further purified on Sephadex LH-20 CC (6 × 55 cm), eluted with water: methanol (2:8) (350 ml), water:methanol (1:9) (500 mL), methanol (450 mL) and methanol:acetone (1:1) (600 mL) to give nine fractions. Subfractions 8 and 9 were combined and further purified on Sephadex LH-20 with methanol (350 mL) and methanol:acetone (1:1) (400 mL) as eluent to yield punicalagins (35 mg). The purification process was monitored by 1D and 2D TLC. Compounds were identified using LCMS based on their retention time, UV absorption pattern, molecular mass, and 1H NMR spectra in relation to standards isolated previously (Bialonska et al., 2009a; Reddy et al., 2007). 2.3. Faecal sample preparation Faecal samples were obtained from three separate individuals. All volunteers were in good health, had not been prescribed antibiotics for at least 6 months before the study and had no history of gastrointestinal disease. Samples were collected, on site, on the day of the experiment and used immediately. A 1:10 (w/v) dilution of the samples in anaerobic phosphate buffer (0.1 M; pH 7.4) was prepared and homogenised in a stomacher for 2 min. Resulting faecal slurries from each individual (i.e. faecal samples were not pooled) were used to inoculate the batch-culture vessels. 2.4. Batch-culture fermentation

Fig. 2. Chemical structure of urolithins.

Batch-culture fermentation vessels (300 mL volume; one vessel per treatment) were sterilised and filled with 135 ml of sterile basal nutrient medium (peptone water (2 g/L), yeast extract (2 g/L), NaCl (0.1 g/L), K2HPO4 (0.04 g/L), KH2PO4 (0.04 g/L), NaHCO3 (2 g/L), MgSO4.7H2O (0.01 g/L), CaCl2.6H2O (0.01 g/L), Tween 80 (2 mL/L),

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

haemin (50 mg/L), vitamin K1 (10 mL/L), L-cysteine (0.5 g/L), bile salts (0.5 g/L), resazurin (1 mg/L) and distilled water) [pH 7.0]. The medium was gassed overnight with O2-free N2. Prior to inoculation of the faecal slurry, the temperature of the basal nutrient medium was maintained at 37 °C by using a circulating water-bath. The pH of the medium was maintained at 6.8, using a pH controller (Electrolab, UK). The batch fermenters were inoculated with 15 mL of faecal slurry (1:10, w/v) and run under anaerobic conditions for a period of 48 h. Samples (7 mL) were collected at five time points (0, 5, 10, 24 and 48 h) for FISH, short chain fatty acid analysis by GC, and ellagitannin metabolism analysis by LC-MS. 2.5. Inoculation of substrate in the batch culture POMx (1.5 mL) and punicalagins (250 mg) were inoculated in stirring batch-culture vessels (one per treatment) containing faecal slurry (1:10, w/v). These amounts were estimated to reflect levels of intake of 1 table spoon of POMx or 250 mL of pomegranate juice. Control experiments incubating POMx and punicalagins in medium without faecal slurry inoculation were also run under anaerobic conditions. Standard prebiotic inulin and starch (1% w/v) were included in the experiment. Fermentations with no added substrate were also included. 2.6. Bacterial enumeration Changes in bacterial populations in media supplemented with pomegranate constituents and standard prebiotics were assessed using FISH with oligonucleotide probes designed to target specific taxonomically relevant regions of 16S rRNA. The probes, commercially synthesised and labelled with the fluorescent dye Cy3, were as follows: EUB338/II/III, for the total number of bacteria (Daims et al., 1999), Bif 164, specific for Bifidobacterium spp. (Langendijk et al., 1995), Lab 158, for Lactobacillus–Enterococcus spp. (Harmsen et al., 1999), Erec 482, for the Clostridium coccoides–Eubacterium rectale group (Franks et al., 1998), and Chis 150 for the C. histolyticum group (Franks et al., 1998). Probe sequences and hybridization conditions are listed in Table 1. The faecal homogenate (375 µL) was fixed in 1.125 µl of 4% (w/v) paraformaldehyde and hybridized with appropriate probes as described previously (Vulevic et al., 2008). The cells were enumerated using the epifluorescent microscope (Brunel Microscopes Ltd, Wiltshire, UK). Microbial counts were expressed as log10 bacterial cells per ml faeces.

177

transferred to a 2 mL Hi Chrom vial (Agilent Technologies, West Lothian, UK). Calibration was performed using standard solutions of acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid, and caproic acid. SCFA were determined by gas–liquid chromatography on an Agilent 5890 II Series GC system (Agilent, Waldbronn, Germany) fitted with an FFAP column (30 m × 0.53 mm, diameter 0.50 μm, J&W Scientific, USA) and a flame-ionization detector. The carrier gas, helium, was delivered at a flow rate of 14 mL/min. The injector, column and detector were set at 280, 140 and 220 °C respectively. The injection volume was 1 μL. Peaks were integrated using a PC running Atlas Lab managing software (Thermo Lab Systems, Mainz, Germany). Fatty acid concentrations were calculated by comparing their peak areas with those of the internal standard and expressed as millimoles per litre.

2.8. Urolithin analysis Undiluted aliquots (1.0 mL) of sample were dispensed into 1.5-mL Eppendorf tubes, acidified with 100 μL of 1 M HCl, vortexed and centrifuged (13,000 ×g, 10 min) to pellet bacteria and other solids. The supernatant was subject to filtration through a reverse phase SepPak Cartridge (Millipore Corp.) previously activated with 10 mL of methanol and 10 mL of distilled water. The cartridge was washed with 1 mL of distilled water and then polyphenol metabolites were eluted with methanol. The methanol fraction (1 mL) was dried under nitrogen flow at room temperature, and the extract redissolved in 250 μL of 1% formic acid in methanol. Peaks representative for urolithins D, C, and A were identified using LC-MS based on their retention time, UV absorption pattern, and molecular mass, in relation to standards previously synthesized (Bialonska et al., 2009b). The LC-MS system consisted of a Waters Micromass® ZQ™ mass spectrometer, Waters 2695 Seperation Module, and Waters 996 Photodiode Array Detector. Mass spectra were recorded in the negative mode. The capillary voltage was 4000/ 3500 V, gas temperature 300 °C, and a 3 × 150 mm analytical column (Phenomenex, Luna 5 μ C18 100 Å) was used. The analyses were performed in the gradient system A — 1% formic acid, B — 1% formic acid in methanol, starting from 100% A for 5 min, 0–60% B for 15 min, and 60–100% B for 15 min. The flow rate was 0.3 ml/min and pressure 900–1500 mm Hg. The elution of metabolites was monitored at 254 nm. The concentration of urolithins was presented as a total ion chromatogram (TIC) peak area.

2.7. Short chain fatty acid analysis

2.9. Statistical analysis

Undiluted aliquots (1.0 mL) of sample were dispensed into 1.5-mL Eppendorf tubes and centrifuged (13,000 × g, 10 min) to pellet bacteria and other solids. The supernatant was acidified with 6 M HCl (3:1 v/v) and incubated at room temperature for 10 min. The mixture was re-centrifuged at 13,000 ×g for 5 min and filtered using a 0.2 µm PVDF filter (Millipore, Cork, Ireland). The internal standard, 2ethylbutyric acid (100 µL), was added to 400 µL of the sample and

Punicalagins and POMx were tested in batch cultures inoculated with faecal samples collected from three individual donors. The log10 numbers of specific bacteria were expressed as mean values with standard deviations. SCFA concentrations were expressed as mean values (mM) with standard deviations. A paired test t was used to test for significant differences at specific time between the control and the treatment vessels.

Table 1 FISH oligonucleotide probes used in this study. Probe name

Target bacterial group/species

Sequence from 5′ to 3′ end

TH⁎ (°C)

Reference

Bif 164 Lab 158 Erec 482 Chis 150 EUB388 EUB388II EUB338III

Bifidobacterium spp. Lactobacillus–Enterococcus spp. Eubacterium rectale–Clostridium coccoides group C. histolyticum group total bacteria total bacteria total bacteria

CATCCGGCATTACCACCC GGTATTAGCACTGTTTCCA CGGTACCTGACTAAGAAGC TTATGCGGTATTAATCTYCCTTT GCTGCCTCCCGTAGGAGT GCAGCCACCCGTAGGTGT GCTGCCACCCGTAGGTGT

50 50 50 50 46 46 46

Langendijk et al. (1995) Harmsen et al. (1999) Franks et al. (1998) Franks et al. (1998) Daims et al. (1999) Daims et al. (1999) Daims et al. (1999)

178

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

3. Results

3.2. Short chain fatty acid concentrations in ellagitannin incubated cultures

3.1. Ellagitannin-induced changes in specific bacterial groups In order to evaluate the changes in bacterial populations in response to pomegranate ellagitannins, we used FISH analysis. This method has been previously used during in vitro and in vivo studies to assess potential prebiotic effects of various substrates. FISH analyses were performed at 0, 5, 10, 24 and 48 h of incubation. POMx significantly (P b 0.05) enhanced the growth of total bacteria, as well as beneficial bacteria Bifidobacterium spp. and the Lactobacillus– Enterococcus group, without effecting the growth of the Clostridium coccoides–Eubacterium rectale group and the C. histolyticum group (P N 0.05) (Table 2). POMx caused a similar enhancement in the growth of total bacteria and bifidobacteria like inulin and starch. However, POMx significantly enhanced (P b 0.05) the growth of lactobacilli, while inulin and starch did not show such effects (Table 2). An increased number of lactobacilli in POMx supplemented media at 10 and 24 h collection time is shown in Fig. 3. The intensity of growth stimulation towards the Lactobacillus–Enterococcus group was determined by the initial composition of faecal inoculum (Fig. 4). An additive growth enhancement was observed upon POMx incubation in the presence of the known prebiotic inulin (Table 2). Hence, bacterial metabolism of pomegranate polyphenols occurs even in the presence of a preferential energy source. The total bacterial number in POMx treated samples increased significantly (P b 0.05) in relation to the control after 10 h of incubation. The statistical significant (P b 0.05) increase in the number of Bifidobacteria spp. in POMx supplemented media was found at the 5 h collection point. No significant effect (P N 0.05) towards the growth of human gut bacteria was observed in media supplemented with punicalagins in the studied concentration (0.2% w/v) (Table 2).

Short chain fatty acid concentrations in media supplemented with pomegranate products were analysed using gas chromatography. POMx incubation significantly (P b 0.05) increased the production of all major short chain fatty acids, acetate, propionate and butyrate, in relation to control (Table 3). Significantly higher (P b 0.05) concentrations of acetate were detected at 10 h collection time. The production of propionate and butyrate was significantly enhanced (P b 0.05) at 24 h of incubation (Table 3). Punicalagins supplementation did not affect the production of short chain fatty acids when compared to control (P N 0.05) (Table 3). 3.3. Metabolism of pomegranate ellagitannins by human faecal microbiota In order to evaluate metabolism of pomegranate ellegitannins by human faecal microbiota, incubation media collected at 0, 5, 10, 24 and 48 h were analysed using LC-MS. The parent punicalagins were not detected in the fermentation media at any collection time. In POMx treated samples the mass peak representative for ellagic acid was detected and gradually disappeared by 10 h of incubation. The dibenzopyranones urolithins A, C and D (Fig. 2) were identified as products of human gut bacterial transformation of POMx components. The transformation of punicalagins yield urolithins C and D (Fig. 5a). Urolithin B, another derivative reported in human systemic circulation after consumption of pomegranate products, was not detected in the fermentation media in this study, neither after supplementation with POMx nor punicalagins at any collection time. The time course study showed tetrahydroxylated urolithin D as the first product of microbial transformation, followed by formation of

Table 2 Log10 number of bacteria per 1 ml of medium supplemented with inulin, starch, POMx, punicalagins (pg), inulin and POMx and without addition of a supplement (control). Values are means with standard deviations. Mean value of the number of specific bacterial group in the treatment group was significantly different from that of control in the corresponding collection time: *p b 0.05, **p b 0.001.

Control

Inulin

Starch

POMx

Punicalagins

Inulin with POMxa

a

Time (h)

Total bacteria

Bifidobacterium spp.

Lactobacillus– Enterococcus group

Eubacterium rectale– Clostridium coccoides group

Clostridium histolyticum group

0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48

9.03 ± 0.06 9.03 ± 0.11 9.03 ± 0.15 9.07 ± 0.15 8.97 ± 0.11 9.00 ± 0.00 9.10 ± 0.10 9.47* ± 0.15 9.60* ± 0.26 9.73* ± 0.30 9.00 ± 0.10 9.20 ± 0.30 9.70* ± 0.26 9.70* ± 0.35 9.73* ± 0.25 9.00 ± 0.20 9.23 ± 0.06 9.53* ± 0.15 9.57* ± 0.15 9.37* ± 0.15 9.03 ± 0.15 9.13 ± 0.11 9.23 ± 0.11 9.27 ± 0.15 9.10 ± 0.15 9.15 9.50 9.65 9.75 9·45

7.87 ± 0.25 7.83 ± 0.06 7.90 ± 0.20 7.90 ± 0.26 7.70 ± 0.17 7.87 ± 0.25 8.57* ± 0.40 8.73* ± 0.23 8.97* ± 0.21 8.93** ± 0.15 7.90 ± 0.20 7.97 ± 0.40 8.63 ± 0.45 9.20* ± 0.17 9.33** ± 0.06 7.83 ± 0.29 8.40* ± 0.26 8.60* ± 0.10 8.80* ± 0.17 8.83** ± 0.15 7.97 ± 0.35 8.13 ± 0.11 8.16 ± 0.15 8.13 ± 0.30 8.23 ± 0.29 7.70 8.90 9.00 9.05 8.95

6.67 ± 0.29 6.83 ± 0.25 6.90 ± 0.30 6.90 ± 0.20 6.97 ± 0.42 6.80 ± 0.35 7.23 ± 0.15 7.10 ± 0.10 7.20 ± 0.36 6.93 ± 0.51 6.83 ± 0.25 7.03 ± 0.32 7.00 ± 0.30 7.10 ± 0.36 7.10 ± 0.61 6.80 ± 0.35 7.63 ± 0.49 7.73* ± 0.42 7.87* ± 0.47 7.87* ± 0.55 6.77 ± 0.21 7.07 ± 0.25 6.93 ± 0.21 7.10 ± 0.36 7.10 ± 0.61 6.50 7.60 7.65 7.65 7.35

8.37 ± 0.15 8.37 ± 0.06 8.17 ± 0.21 8.27 ± 0.15 7.90 ± 0.35 8.30 ± 0.20 8.43 ± 0.06 8.47 ± 0.11 8.77 ± 0.30 8.47 ± 0.30 8.33 ± 0.15 8.47 ± 0.06 8.87 ± 0.35 8.83 ± 0.40 8.47* ± 0.21 8.47 ± 0.06 8.33 ± 0.21 8.47 ± 0.25 8.47 ± 0.11 8.00 ± 0.20 8.37 ± 0.06 8.27 ± 0.15 8.00 ± 0.10 8.00 ± 0.10 7.40 ± 0.60 8.45 8.30 8.45 8.65 8.20

6.80 ± 0.17 6.80 ± 0.20 6.83 ± 0.35 6.73 ± 0.21 6.77 ± 0.32 6.80 ± 0.20 7.13 ± 0.25 7.00 ± 0.20 6.90 ± 0.36 6.63 ± 0.25 6.73 ± 0.11 7.07 ± 0.21 6.83 ± 0.25 6.87 ± 0.35 6.77 ± 0.32 6.77 ± 0.15 6.17 ± 0.46 6.83 ± 0.25 7.07 ± 0.11 6.87 ± 0.07 6.75 ± 0.21 6.80 ± 0.17 6.67 ± 0.15 6.63 ± 0.15 6.60 ± 0.26 6.75 7.25 7.25 7.20 6.55

Numbers represent mean value of two experiments.

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

179

Fig. 3. FISH analysis of Lactobacillus–Enterococcus group in batch culture media supplemented with inulin, POMx and with no additives (control) at 0, 10 and 24 h of incubation.

derivatives with a decreasing number of phenolic hydroxy groups: the trihydroxy analogue urolithin C and dihydroxylated urolithin A (Fig. 5b). The rate of ellagitannin transformation varied in batch cultures inoculated with faecal samples obtained from different donors (Fig. 5b). In batch culture 3, urolithin D was detected as the first product of transformation at the 10 h collection time, followed by urolithin C at 24 h and urolithin A at 48 h (Fig. 5b). In the batch culture inoculated with faecal sample 2, both urolithins D and C were detected at 10 h of incubation. After 24 h, urolithin D was absent but both urolithins A and C were present (Fig. 5b). In batch 1, urolithins D and C were detected for the first time at 24 h collection time. In the

Table 3 Concentrations of major SCFA during 48 h fermentation in media supplemented with inulin, starch, POMx, punicalagins (pg), inulin and POMx and without addition of a supplement (control). Values are means with standard deviations. Mean value of the SCFA concentration in the treatment group was significantly different from that of control in the corresponding collection time: *p b 0.05, **p b 0.001. Time (h) Control

Inulin

Starch

POMx

Punicalagins

Inulin with POMxa

Fig. 4. Difference in log10 number of Lactobacillus–Enterococcus spp. bacteria per 1 ml of medium supplemented with POMx and without addition of a supplement (control) in batch cultures inoculated with faecal samples of three different donors.

a

0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48 0 5 10 24 48

Acetate 0.1 ± 0.2 2.3 ± 0.8 4.2 ± 0.6 6.3 ± 0.9 8.2 ± 1.5 0.2 ± 0.2 12.0 ± 10.8 24.4* ± 11.3 29.5* ± 8.2 36.6** ± 5.0 0.1 ± 0.1 8.1 ± 5.1 20.3* ± 6.9 38.9* ± 5.0 43.1* ± 10.3 0.6 ± 0.1 9.3 ± 5.8 16.3* ± 3.5 23.2* ± 2.8 25.6* ± 3.5 0.3 ± 0.2 2.6 ± 0.4 3.6 ± 0.7 6.3 ± 1.0 7.2 ± 1.0 0·4 22·4 34·8 40·9 46·3

Numbers represent mean value of two experiments.

Propionate

Butyrate

0.0 ± 0.0 0.6 ± 0.1 1.1 ± 0.3 1.6 ± 0.6 2.0 ± 0.3 0.0 ± 0.0 2.0 ± 2.1 6.6 ± 5.4 9.4* ± 5.4 11.5* ± 3.7 0.0 ± 0.0 2.0 ± 1.8 4.4 ± 2.2 6.6* ± 2.4 7.2* ± 2.2 0.0 ± 0.0 2.8 ± 2.9 6.5 ± 2.8 11.0* ± 2.7 11.9* ± 3.7 0.0 ± 0.0 0.6 ± 0.2 1.1 ± 0.3 1.8 ± 0.7 1.9 ± 0.6 0·0 3·6 10·8 19·5 22·9

0.0 ± 0.0 0.0 ± 0.0 0.5 ± 0.2 1.2 ± 0.3 1.9 ± 0.2 0.0 ± 0.0 0.4 ± 0.3 2.8 ± 1.9 5.7* ± 1.3 8.5* ± 3.7 0.0 ± 0.0 1.2 ± 1.8 3.1 ± 2.7 5.3* ± 2.4 6.5* ± 2.5 0.0 ± 0.0 0.1 ± 0.1 1.0 ± 0.8 2.8* ± 0.8 3.6* ± 0.4 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.2 0.8 ± 0.5 1.6 ± 0.4 0·0 0·0 0·9 4·7 6·6

180

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

Fig. 5. a. Concentration of urolithins D (UD), C (UC) and A (UA) in fermentation media supplemented with POMx and punicalagins at 48 h collection time. Values are means with standard deviations represented by vertical bars. b. Concentration of urolithins D (UD), C (UC) and A (UA) at 0, 5, 10, 24 and 48 h in fermentation media supplemented with POMx (1% v/v) and inoculated with faecal slurry from three different donors.

sample collected at 48 h urolithin D was not present, but urolithin C and trace amounts of urolithin A were indeed detected (Fig. 5b). The intensity of urolithins production also varied between individual fermentations. A higher concentration of urolithins was detected in fermentation media inoculated with faecal bacteria from donors 2 and 3. The rate of bacterial transformation of ellagitannins into urolithins was the lowest in the culture inoculated with faecal sample obtained from donor 1.

4. Discussion In the present study, the pomegranate ellagitannin punicalagins and pomegranate by-product (POMx) were tested for their influence on the human gut bacteria. POMx significantly enhanced the growth of bifidobacteria and lactobacilli, bacterial groups associated with various health benefits at the cellular and systemic levels. Owing to their ability to generate short chain fatty acids these bacteria have been linked to an inhibition of pre-neoplastic proliferation and acceleration of conversion of cholesterol into bile acids (Gibson et al., 1995; Rastall et al., 2005). Indeed, the enhanced growth of bacteria after POMx treatment was reflected in the increased concentrations of the major short chain fatty acids, i.e., acetate, propionate and butyrate in culture media. POMx produced a similar enhancement in the growth of total bacteria and bifidobacteria like inulin and starch. However, POMx also enhanced the growth of lactobacilli, while inulin and starch did not show such effects. The correlation between bacterial composition of faecal inoculum and the intensity of effects towards lactobacilli, suggest the involvement of specific strains in the decomposition of POMx oligomers and release of glucose and free units of ellagic and gallic acids. For example, an

increased activity of α-glucosidase was found in Bifidobacterium breve grown in POMx supplemented medium (unpublished data). POMx did not influence the growth of the Clostridium coccoides– Eubacterium rectale group and the C. histolyticum group. The lack of growth enhancement is specially relevant in case of C. histolyticum group, composed of some proteolytic bacteria, because their metabolism may contribute to the progression of colorectal cancer and inflammatory bowl diseases (Gibson, 2008). In addition, by inducing the growth of probiotic bacteria, POMx may inhibit the ability of potential pathogenic species to colonise the colon (Gibson, 2008; Onoue et al., 1997). To evaluate their influence on faecal microbiota, punicalagins were supplemented into media at an equimolar concentration based on their predicted content in POMx. This concentration can also be achieved in the intestines after consumption of pomegranate juice. In general, no significant effect towards the growth of human gut bacteria or production of short chain fatty acids, were observed. Based on these in vitro studies, intake of large amounts of products containing punicalagins should not disturb the ratio of beneficial human gut bacteria. Previously, punicalagins showed selective inhibitory effects towards pure isolates of pathogenic bacteria, e.g. E. coli, P. aeruginosa (Reddy et al., 2007) and selected strains of Clostridium sp. (Bialonska et al., 2009a). This likely provides protection against overgrowth of pathogenic strains in the intestines. Since punicalagins did not increase the growth of faecal microbiota in the batch culture, the effects of POMx on the faecal bacteria are probably attributed to the abundance of the oligomers composed of 2–10 repeating units of gallic acid, ellagic acid, and glucose in different combinations. Upon faecal bacterial incubation, POMx and punicalagins have been transformed to the dibenzopyranone-type urolithins. Urolithins were at first detected as metabolites present in systemic circulation after consumption of pomegranate products (MertensTalcott et al., 2006; Seeram et al., 2006), and later were confirmed as products of intestinal microbial transformations (Cerda et al., 2004; Ito et al., 2008; Larrosa et al., 2006b). In the present study, we report for the first time urolithins C and D as products of ellagitannin transformation by human faecal microbiota. Production and distribution of urolithins were investigated in vivo in Iberian Pigs fed with an ellagitannin rich diet (Espin et al., 2007). Significant amounts of urolithins D and C, and trace amounts of urolithin A were detected in the jejunum. Hence, bacteria present in the small intestines are able to transform ellagitannins to a number of metabolites (Espin et al., 2007). Since urolithin B was not detected in the small intestines, it suggests that the bacterial metabolism of ellagitannins continues in the colon, and culminates with the formation of urolithin B as the final product (Espin et al., 2007). The distribution of urolithins in the digestive track also points to urolithin D as the first product of microbial transformation of ellagic acid, and subsequent modifications lead to intermediates with a decreasing number of phenolic hydroxy groups: urolithin C (3,7,8-trihydroxydibenzopyranone), urolithin A (3,8-dihydroxydibenzopyranone), and urolithin B (3hydroxydibenzopyranone) (Espin et al., 2007). The same pathway of urolithins formation was confirmed in the time course profile of batch culture fermentation by human faecal microbiota in the present study. The rate and efficiency of transformation of POMx ellagitannins into urolithins differed in individual batch cultures inoculated with faecal slurry obtained from different donors. The first analogues, urolithins D and C were detected in media between 10 and 24 h of incubation. Urolithin A was formed between 24 and 48 h, which is in agreement with the appearance of this metabolite in plasma found in human volunteers (Cerda et al., 2004; Mertens-Talcott et al., 2006). Therefore, the individual composition of intestinal microbiota determines the efficiency of ellagitannin transformation into bioavailable urolithins and their concentration in systemic circulation.

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

Urolithin B, was not detected in this study in the fermentation medium, neither after supplementation with POMx nor punicalagins, at any collection time. This can be explained in terms of insufficient duration of the fermentation process, since this compound is the last product in the urolithin formation cascade. By transformation of ellagitannins into urolithins, intestinal microbiota are able to modify bioavailability and bioactivity of parent molecules. Consequently, bacteria significantly contribute to the health beneficial action of pomegranate products. For example, pomegranate polyphenols exhibited high antioxidant potency attributed to multiple phenolic hydroxy groups in the HHDP and gallagyl moieties with potential to form o- or p-quinones (Reddy et al., 2007). Regular consumption of these compounds is likely to increase the antioxidant capacity of the gastrointestinal track. This may be of special significance in the large intestines, where oxidative stress is generated during inflammatory bowel diseases (Garsetti et al., 2000). The antioxidant activity of parent ellagitannins and gut microbial metabolites were compared after evaluation in a cell based assay (Bialonska et al., 2009b). Urolithins C and D, present in significant amounts in the small intestines, inhibited ROS generation with higher potency than original ellagic acid and punicalagins (Bialonska et al., 2009b). Urolithin A, the major metabolite present in systemic circulation exhibited a relatively weak antioxidant action in relation to ellagitannins. Despite this, the antioxidant IC50's of 13 μM was still in the range of the plasma concentrations of urolithin A (Bialonska et al., 2009b). In another study, pomegranate extract and urolithin A were tested for anti-inflammatory properties in a colitis rat model. Urolithin A accounted for more effective protection against colon inflammation. However, the rate of ellagitannin transformations to urolithins significantly decreased in inflamed colon. In consequence, non-metabolized ellagitannin fraction is likely the major antiinflammatory factor in subjects suffering from inflammation (Larrosa et al., 2009). In conclusions, pomegranate by-product enhanced the growth of bifidobacteria and lactobacilli as well as production of short chain fatty acids in media inoculated with human faecal microflora. We are aware that in vitro investigation using a batch culture model, do not reflect the effects of pomegranate ellagitannins on human gut bacteria. However, similar growth enhancement was detected in faeces of rats fed with pomegranate extract (Larrosa et al., 2009). Pomegranate was also confirmed as a potent antiobesity remedy (Lei et al., 2007), which is likely achieved by stimulation of probiotic bacteria. In addition, many health beneficial effects of pomegranate have been attributed to urolithins, products of their microbial intestinal transformation. Therefore, human studies on potential of pomegranate by-product to act as prebiotic would be highly valuable.

Acknowledgments We thank POM Wonderful, Los Angeles, for financial support. We greatly appreciate the assistance of Dr Gemma E. Walton in batch culture systems and photography.

References Adams, L.S., Seeram, N.P., Aggarwal, B.B., Takada, Y., Sand, D., Heber, D., 2006. Pomegranate juice, total pomegranate ellagitannins, and punicalagin suppress inflammatory cell signalling in colon cancer cells. Journal of Agriculture and Food Chemistry 54, 980–985. Ahn, Y.J., Lee, C.O., Kweon, J.H., Ahn, J.W., Park, J.H., 1998. Growth-inhibitory effects of Galla Rhois-derived tannins on intestinal bacteria. Journal of Applied Microbiology 84, 439–443. Bialonska, D., Kasimsetty, S.G., Shrader, K.K., Ferreira, D., 2009a. The effect of pomegranate (Punica granatum L.) by-product and ellagitannins on the growth of human gut bacteria. Journal of Agriculture and Food Chemistry 57, 8344–8349.

181

Bialonska, D., Kasimsetty, S.G., Khan, S.I., Ferreira, D., 2009b. Urolithins, microbial metabolites of pomegranate ellagitannis, exhibit potent antioxidant activity in a cell based assay. Journal of Agriculture and Food Chemistry 57, 10181–10186. Cerda, B., Espin, J.C., Parra, S., Martinez, P., Tomas-Barberan, F.A., 2004. The potent in vitro antioxidant ellagitannins from pomegranate juice are metabolised into bioavailable but poor antioxidant hydroxyl-6H-dibezopyran-6-one derivatives by the colonic microflora of healthy humans. European Journal of Nutrition 43, 205–220. Daims, H., Brühl, A., Amann, R., Schleifer, K.H., Wagner, M., 1999. The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Systematic and Applied Microbiology 22, 434–444. Espin, J.C., Gonzalez-Barrio, R., Cerda, B., Lopez-Bote, C., Rey, A.I., Tomas-Barberan, F.A., 2007. Iberian Pig as a model to clarify obscure points in the bioavailability and metabolism of ellagitannins in humans. Journal of Agriculture and Food Chemistry 55, 10476–10485. Franks, A.H., Harmsen, H.J.M., Raangs, G.C., Jansen, J., Schut, F., Welling, G.W., 1998. Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16 S rRNA-targeted oligonucleotide probes. Applied and Environmental Microbiology 64, 3336–3345. Garsetti, M., Pellegrini, N., Baggio, C., Brighenti, F., 2000. Antioxidant activity in human faeces. British Journal of Nutrition 84, 705–710. Gibson, G.R., 2008. Prebiotics as gut microflora management tools. Journal of Clinical Gastroenterology 42, S75–S79. Gibson, G.R., Beatty, E.R., Wang, X., Cummings, J.H., 1995. Selective stimulation of bifidobacteria in the human colon by oligofructose and inulin. Gastroenterology 108, 975–982. Gil, M.I., Tomas-Barberan, F.A., Hess-Pierce, B., Holcroft, D.M., Kader, A.A., 2000. Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. Journal of Agriculture and Food Chemistry 48, 4581–4589. Harmsen, H.J.M., Elfferich, P., Schut, F., Welling, G.W., 1999. A 16 s RNA-targeted probe for the detection of lactobacilli and enterococci in faecal samples by fluorescent in situ hybridisation. Microbial Ecology in Health and Disease 11, 3–11. Hord, N.G., 2008. Eukaryotic-microbiota crosstalk: potential mechanisms for health benefits of prebiotics and probiotics. Annual Review of Nutrition 28, 215–231. Ito, H., Iguchi, A., Hatano, T., 2008. Identification of urinary and intestinal bacterial metabolites of ellagitannin geraniin in rats. Journal of Agriculture and Food Chemistry 56, 393–400. Langendijk, P.S., Schut, F., Jansen, G.J., Raangs, G.C., Kamphuis, G.R., Wilkinson, M.H.F., Welling, G.W., 1995. Quantitative fluorescence in situ hybridization of Bifidobacterium spp. with genus-specific 16 s ribosomal-RNA-targeted probes and its application in fecal samples. Applied and Environmental Microbiology 61, 3069–3075. Larrosa, M., Gonzalez-Sarrias, A., Garcia-Conesa, M.T., Tomas-Barberan, F.A., Espin, J.C., 2006a. Urolithins, ellagic acid-derived metabolites produced by human colonic microflora, exhibit estrogenic and antiestrogenic activities. Journal of Agriculture and Food Chemistry 54, 1611–1620. Larrosa, M., Tomas-Barberan, F.A., Espin, J.C., 2006b. The hydrolysable tannin punicalagin releases ellagic acid which induces apoptosis in human colon adenocarcinoma Caco-2 cells by using the mitochondrial pathway. Journal of Nutritional Biochemistry 17, 611–625. Larrosa, M., González-Sarrías, A., Yáñez-Gascón, M.J., Selma, M.V., Azorín-Ortuño, M., Toti, S., Tomás-Barberán, F., Dolara, P., Espín, J.C., 2009. Anti-inflammatory properties of a pomegranate extract and its metabolite urolithin-A in a colitis rat model and the effect of colon inflammation on phenolic metabolism. Journal of Nutritional Biochemistry. doi:10.1016/j.jnutbio.2009.04.012. Lee, H.C., Jenner, A.M., Low, C.S., Lee, Y.K., 2006. Effect of tea phenolics and their aromatic fecal bacterial metabolites on intestinal microbiota. Research in Microbiology 157, 876–884. Lei, F., Zhang, X.N., Wang, W.D., Xing, M., Xie, W.D., Su, H., Du, L.J., 2007. Evidence of anti-obesity effects of the pomegranate leaf extract in high-fat diet induced obese mice. International Journal of Obesity 31, 1023–1029. Mertens-Talcott, S.U., Jilma-Stohlawetz, P., Rios, J., Hingorani, L., Derendorf, H., 2006. Absorption, metabolism, and antioxidant effects of pomegranate (Punica granatum L.) polyphenols after ingestion of a standardized extract in healthy human volunteers. Journal of Agriculture and Food Chemistry 54, 8956–8961. Onoue, M., Kado, S., Sakaitani, Y., Uchida, K., Morotomi, M., 1997. Specific species of intestinal bacteria influence the induction of aberrant crypt foci by 1, 2dimethylhydrazine in rats. Cancer Letters 113, 179–186. Parkar, S.G., Stevenson, D.E., Skinner, M.A., 2008. The potential influence of fruit polyphenols on colonic microflora and human gut health. International Journal of Food Microbiology 124, 295–298. Rastall, R.A., Gibson, G.R., Gill, H.S., Guarner, F., Klaenhammer, T.R., Pot, B., Reid, G., Rowland, I.R., Sanders, M.E., 2005. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and synbiotics to enhance human health: an overview of enabling science and potential applications. FEMS Microbiology Ecology 52, 145–152. Reddy, M.K., Gupta, S.K., Jacob, M.R., Khan, S.I., Ferreira, D., 2007. Antioxidant, antimalarial and antimicrobial activities of tannin-rich fractions, ellagitannins and phenolic acids from Punica granatum L. Planta Medica 73, 461–467. Seeram, N.P., Adams, L.S., Henning, S.M., Niu, Y., Zhang, Y., Nair, M.G., Heber, D., 2005. In vitro antiproliferative, apoptotic and antioxidant activities of punicalagin, ellagic acid and a total pomegranate tannin extract are enhanced in combination with other polyphenols as found in pomegranate juice. Journal of Nutritional Biochemistry 16, 360–367.

182

D. Bialonska et al. / International Journal of Food Microbiology 140 (2010) 175–182

Seeram, N.P., Henning, S.M., Zhang, Y., Suchard, M., Li, Z., Heber, D., 2006. Pomegranate juice ellagitannin metabolites are present in human plasma and some persist in urine for up to 48 hours. Journal of Nutrition 136, 2481–2485. Seeram, N.P., Lee, R., Heber, D., 2008. Bioavailability of ellagic acid in human plasma after consumption of ellagitannins from pomegranate (Punica granatum L.) juice. Clinica Chimica Acta 348, 63–68. Tzounis, X., Vulevic, J., Kuhnie, G.G.C., George, T., Leonczak, J., Gibson, G.R., Kwik-Uribe, C., Spencer, J.P.E., 2008. Flavanol monomer-induced changes to the human faecal microflora. British Journal of Nutrition 99, 782–792.

Vulevic, J., Drakoularakou, A., Yaqoob, P., Tzortzis, G., Gibson, G.R., 2008. Modulation of the faecal microflora profile and immune function by a novel trans- galactooligosaccharides mixture (B-GOS) in healthy elderly volunteers. American Journal of Clinical Nutrition 88, 1438–1446.