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Bioconversion of daidzein to equol by Bifidobacterium breve15700 and Bifidobacterium longum BB536

Salma Elghalia, Shuhaimi Mustafaa,*, Mehrnoush Amidb, Mohd Yaizd ABD Manapb,Amin Ismailc, Faridah Abasd

aDepartment of Microbiology, Faculty of Biotechnology and Biomolecular Sciences, University Putra Malaysia (UPM), 43400 Serdang,

Selangor, MalaysiabDepartment of Food Technology, Faculty of Food Science and Technology, University Putra Malaysia (UPM), 43400 Serdang, Selangor,

MalaysiacDepartment of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, University Putra Malaysia (UPM), 43400 Serdang, Selangor,

MalaysiadDepartment of Food Science, Faculty of Food Sciences and Technology, University Putra Malaysia (UPM), 43400 Serdang, Selangor, Malaysia

A R T I C L E I N F O A B S T R A C T

Article history:

Received 11 January 2012

Received in revised form

25 April 2012

Accepted 25 April 2012

Available online 28 May 2012

Keywords:

Bifidobacterium spp

Phytoestrogens

Isoflavones

Daidzein

Equol

1756-4646/$ - see front matter � 2012 Elsevihttp://dx.doi.org/10.1016/j.jff.2012.04.013

* Corresponding author: Tel.: +60 03 8946 671E-mail address: shuhaimi@biotech.upm.e

Bifidobacteria species were incubated anaerobically with daidzein in Brain Heart Infusion

broth at 37 �C for 96 h. Equol production started during the first 6 h incubation with Bifido-

bacterium breve ATCC 15700 (B. breve) and Bifidobacterium longum BB536 (B. longum). The pH

decreased during incubation of the bacteria with daidzein more than that in its absence.

However, there was no significant difference in the growth of bifidobacteria strains when

incubated in the culture medium with or without daidzein. The production of lactic and

acetic acids after 96 h incubation with daidzein for B. breve was 5.53 and 8.83 mmol l�1,

respectively, while for B. longum was 6.86 and 7.20 mmol l�1, respectively. Thus, probiotic

bacteria were able to produce equol from daidzein, hence dietary supplementation

with equol may offer an important approach to provide all consumers with the health-

promoting benefits of this metabolite.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Phytoestrogens are known as estrogen-like compounds found

in legumes, particularly soybeans, and other plants such as

red clover. Phytoestrogens are classified into three main

groups, of isoflavones, lignans, and coumestans. Moreover,

they are structurally similar to mammalian estrogen (17-b-

estradiol) (Setchell et al., 1980) and functionally mimic the

estradiol in the human body (Setchell & Cassidy, 1999;

Setchell, Zimmer-Nechemias, Cai, & Heubi, 1998). Isoflavones

have potential protective effects on breast cancer, prostate

cancer, cardiovascular disease, and menopausal symptoms

er Ltd. All rights reserved

1.du.my (S. Mustafa).

(Adlercreutz, 2002; Duncan, Phipps, & Kurzer, 2003). Studies

have established that human intestinal bacteria play a role

in isoflavone metabolism (Setchell, Borriello, Hulme, Kirk, &

Axelson, 1984). Host health is affected by the metabolism of

bioactive food components by gut microbiota. Moreover,

some functional food components influence the growth

and/or metabolic activity of the intestinal bacteria and, thus

their composition and function (Campbell, Fahey, & Wolf,

1997; Gibson, McCartney, & Rastall, 2005). Therefore, the

intestinal bacteria are both a target for nutritional interven-

tion to improve human health, and also influencing the bio-

logical activity of other food compounds acquired orally.

.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5 737

Bifidobacteria are absolutely useful human gut microbiota

(Gibson, 1998) and thus they are termed ‘probiotic bacteria’

(O’Sullivan, 2001). These bacteria possess high b-glucosidase

activity (Gibson, 1998; O’Sullivan, 2001), essential for the ini-

tial hydrolysis of the soy isoflavone glucosides and for subse-

quent absorption of the bioactive aglycones (Turner,

Thomson, & Shaw, 2003). These probiotic bacteria can be

added to the food matrix as live cultures such as in yoghurt

in order to increase their numbers in the gastrointestinal tract

(Cummings & Macfarlane, 1997). Developed gastrointestinal

microbiota composition is quite steady, diet can, to a large ex-

tent, alter the metabolic activity of certain bacterial popula-

tion (Hentges, 1980; Parodi, 1999).

Among isoflavones, daidzein (DAI) and genistein (GEN) are

the most common isoflavones studied (Reinli & Block, 1996). It

has been reported that DAI is converted either to equol (EQL)

or O-desmethylangolensin (O-DMA) via dihydrodaidzein

(DHD) (Kelly, Nelson, Waring, Joannou, & Reeder, 1993). Only

30–40% of the human population can biologically produce

EQL by the activity of the intestinal bacteria (Sathyamoorthy

& Wang, 1997; Setchell & Adlercreutz, 1988). This fact has

been explained by many previous published findings, which

reported that the dissimilarities in population of colonic bac-

teria (Atkinson, Berman, Humbert, & Lampe, 2004; Rafii, Da-

vis, Park, Heinze, & Beger, 2003; Setchell et al., 2002) and

dietary variation in human (Decroos, Vanhemmens, Cattoir,

Boon, & Verstraete, 2005; Setchell et al., 2002) are responsible

for isoflavones conversion. EQL has a stronger estrogenic

activity than DAI and O-DMA (Sathyamoorthy & Wang,

1997). It is known that humans who are able to produce EQL

from DAI (equol producers) face a lower threat of mounting

breast and prostate cancer than non-equol producers (Akaza

et al., 2004). It was found that a continuous 7-day supplement

of equol decreases ventral prostate weight in rats, suggesting

that equol may be efficient in reducing the risk of and to some

extent prevention of prostate cancer (Lund et al., 2004).

Although extensive research has been performed to find

out whether a single bacterium is able to produce EQL from

isoflavones or not, only a few findings have reported that ‘‘a

single bacterium has the ability to produce EQL from DAI’’.

Hur et al. (2002) reported that the isolation of Clostridium

sp. HGH6 from human faecal can metabolise DAI to DHD An-

other finding reported that isolation of human intestinal bac-

terium Eggerthella sp. Julong 732 produces S-equol from

dihydrodaidzein (Wang, Hur, Lee, Kim, & Kim, 2005). Isolation

of bacterium strain Lactobacillus sp. Niu-O16 that can pro-

duce DHD from DAI was also reported (Wang, Shin, Hur, &

Kim, 2005). In a previous study, Lactococcus 20-92 from hu-

man faeces that produced EQL from DAI was isolated (Shi-

mada et al., 2010). Adlercreutzia equolifaciens directly

produces EQL from DAI as single strain (Maruo, Sakamoto,

Ito, Toda, & Benno, 2008) and Asaccharobacter celatus AHU

1763, which is able to transfer DAI to EQL via DHD was iso-

lated from human faeces (Thawornkuno, Tanaka, Sone, &

Asano, 2009).

Due to the important function of bifidobacteria as probi-

otic bacteria, and because some studies (Raimondi, De Lucia,

Amaretti, Leonardi, & Pagnoni, 2009) have reported that bifi-

dobacterial species are not able to produce equol, this study

hypothesised that bifidobacterial species were able to produce

equol from daidzein derived from the facts that there is diver-

sity among bifidobacterial species and among strains of the

same species. This study – intended to examine the probiotic

bacteria (Bifidobacterium spp.) for equol productionand to

study the effect of daidzein on the growth of probiotic

bacteria.

2. Materials and methods

2.1. Chemicals

Daidzein and equol were purchased from Sigma Chemical Co.

(St. Louis, MO, USA). All chemicals (acetonitrile, methanol,

diethylether, and ethanol) were purchased from Merck,

Darmstadt, Germany.

2.2. Bacterial species and storage condition

Bifidobacterium breve ATCC 15700 and Bifidobacterium longum

(BB536) were obtained from Biotechnology and Functional

Food Laboratory (Faculty of Food Science and Technology,

UPM, Malaysia). The purity of the bacterial species was

checked with Gram staining and the stock culture was propa-

gated and stored at �80 �C in 40% glycerol for further use.

2.3. Preparation of inoculums for equol production

Bifidobacterium strains were activated by inoculation into

Brain Heart Infusion (BHI) broth medium at 37 �C in anaerobic

chamber for 24 h. One millilitre of each of the pre-cultured

bacterial strain was inoculated into 10 ml of BHI broth con-

taining 200 ll of 40 mM daidzein (DAI). Bacterial cultures were

incubated anaerobically at 37 �C and 1 ml samples were taken

at 0, 6, 12, 18, and 24 h, and continued in every 24 h thereafter

for a total of 96 h. Samples were taken for HPLC analysis to

determine the production of equol.

2.4. HPLC analysis

2.4.1. Sample preparationAfter centrifugation of the cultured medium, the supernatant

was extracted three times with diethyl ether (Decroos et al.,

2005) at a ratio of 1:1 (v/v). Diethyl ether was evaporated

and the remaining extract was redissolved in ethanol and fil-

tered using a 0.45 lm membrane filter.

2.4.2. HPLC analysisEquol was detected according to the method of Wang, Kim,

Kang, Kim, & Hur, 2007 with modifications. Twenty microli-

tres of sample were injected into HPLC (Model CO-2065 JASCO

Corporation Hachioji, Tokyo, Japan) equipped with C18 re-

versed-phase column (25 cm · 4.5 cm · 5 mM) (Ascentis–

Supelco, Sigma–Aldrich Co. LLC. L, USA), diode array ultravio-

let (UV) visible detector, vacuum degasser, and thermostati-

cally controlled column compartment. Column temperature

was set at 27 �C. HPLC gradient elution was composed of

10% acetonitrile solution in water (solution A) and 90% aceto-

nitrile solution in water (solution B). The elution program was

as follows: solution B was run at 30% for 15 min, linearly

Fig. 1 – (A): High performance liquid chromatography profile

for isoflavones isomers standards mix. The peaks are:

daidzein, 6.827 min, equol, and 10.227 min. (B): Reversed-

phase HPLC chromatograms showing the compounds found

in the extract of the BHI medium supplemented with

daidzein and fermented with B. breve. Peaks are daidzein;

6.84 min, equol: 10.013 min. (C): Reversed-phase HPLC

chromatograms showing the compounds found in the

extract of the BHI medium supplemented with daidzein and

fermented with B. longum BB536. Peaks are daidzein;

6.867 min, equol: 10.053 min.

738 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5

increased to 50% for 10 min, and then linearly increased to

70% for 5 min. The flow rate was at 1 ml/min. A diode array

UV–visible detector was set at 270 nm. UV spectra and reten-

tion times of the metabolites produced from daidzein by bac-

teria were compared with those of the standard compounds

daidzein and equol in HPLC chromatograms.

2.5. Enumeration of viable bacteria

Enumeration of viable population of Bifidobacterium strains at

different time intervals (6, 12, 18, 24, 48, 72, and 96 h) was

done using spread plate method. The 1 ml fermentation broth

was serially diluted up to 10-fold in peptone saline followed

by spreading on BHI agar (Oxoid Ltd., Hampshire, England).

The plates were anaerobically incubated using Anaerobic� A

at 37 �C for 48 h. The growth was calculated as log10 Colony

Forming Unit (CFU) per ml. Colony counts between 30 and

300 were enumerated. The experiment was done in duplicate.

2.6. Determination of pH and organic acids duringfermentation

The pH of the medium was measured every 6 h during the

first 24 h of incubation period and then every 24 h up to

96 h. The pH was monitored at 25 �C after calibrating with

pH 4.0, 7.0, and 9.21 standard buffers using a microprocessor

pH metre (Model Delta 320, Mettler-Toledo Instruments-Ltd.,

Shanghai, China). The supernatant of the fermentation med-

ium was analysed to determine the formation of organic acids

during incubation.

2.7. Supernatant preparation

Two millilitres of the fermented medium were centrifuged at

1450g for 30 min at 25 �C. The supernatant was filtered

through a 0.22 lm syringe membrane into HPLC vials and

stored at �20 �C for analysis of organic acids. Profiles of or-

ganic acids were analysed by high-performance liquid chro-

matography (HPLC) (Shimadzu LC-10AS Liquid

Chromatograph, Kyoto, Japan) with a Shimadzu SPD-10AV

UV–VIS detector. An organic column packed with 9 lm of

polystyrene divinylbenzene ion exchange resin (Aminex

HPX-87H, 300 mm · 7.8 mm, Bio-Rad Laboratories, Hercules,

CA, USA) and maintained at 65 �C was used. The UV detector

was set at 220 nm and the mobile phase was 0.0045 M sulphu-

ric acid with a flow rate of 0.6 ml/min (Liong & Shah, 2006).

The HPLC system was calibrated using standards for lactic

and acetic acids.

2.8. Liquid chromatography mass spectrometry analysis(LC/MS)

LC-MS analysis to demonstrate the changing pathway for

daidzein. Analyses were performed, using Thermo Electron,

Finnigan LCQ surveyor MS detector with Electron, Finnigan

LCQ Surveyor UV detector and Thermo Electron Finnigan Sur-

veyor LC System (San Jose, CA, US) and Hypersil gold

(150 · 2.1 mm I.D., 5 lm) column (Thermo-Scientific, Hudson,

NH, USA). Isoflavones were eluted with a mobile phase of

solvent A (0.5% acetic acid in H2O) and solvent B (100% aceto-

nitrile) at a flow rate of 100 ll/ min.

3. Statistical analysis

Data analysis was done using SPSS version 16. Differences be-

tween means for daidzein and equol concentrations at different

Table 1 – Concentration of equol (mmol l�1) in Brain Heart Infusion medium supplemented with 200 ml of daidzein(40 mM/ml) for the period of fermentation with Bifidobacterium breve 15700 and Bifidobacterium longum BB536.

Incubation period (h) Changes in daidzein and equol concentrations

Daidzein Equol

Control B. breve B. longum Control B. breve B. longum

0 40.37 ± 19.0a 40.35 ± 0.41a 40.38 ± 0.18a nd nd nd

6 40.44 ± 0.54a 37.91 ± 0.12b 38.21 ± 0.18b nd 0.016 ± 1.20a 0.026 ± 2.03a

12 40.47 ± 0.64a 34.09 ± 0.62b 34.51 ± 0.77b nd 0.035 ± 2.87a 0.089 ± 8.25a

18 40.70 ± 14.0a 29.96 ± 0.06b 30.55 ± 0.28b nd 0.059 ± 3.56a 0.117 ± 9.35a

24 40.50 ± 56.0a 27.14 ± 0.33b 27.03 ± 0.48b nd 0.105 ± 33.20b 0.171 ± 16.50b

48 40.60 ± 32.0a 24.66 ± 0.4b 23.5 ± 0.72b nd 0.160 ± 24.06b 0.220 ± 7.90b

72 40.50 ± 52.0a 21.97 ± 0.72b 19.32 ± 1.19b nd 0.220 ± 8.95b 0.243 ± 7.27b

96 40.20 ± 36.0a 11.70 ± 1.37b 15.81 ± 0.61b nd 0.291 ± 31.67c 0.314 ± 31.16c

Values are means ± STD for triplicate independent experiments. Different superscript letters in the same column indicate significant (p < 0.05)

differences between means. nd = Not detected-not determined.

240.0 240.5 241.0 241.5 242.0 242.5 243.0 243.5 244.0m/z

68

70

72

74

76

78

80

82

84

86

88

90

92

94

96

98

100

Rel

ativ

e Ab

unda

nce

241.46

243.56

250.0 250.5 251.0 251.5 252.0 252.5 253.0 253.5 254.0 254.5 255.0m/z

0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

650000

Inte

nsity

253.59

251.38

254.28

1

2

2

3

1

A

B

Fig. 2 – Representative LC MS chromatogram of equol and daidzein standards using negative acquisition mode. (A) Peak 1

equol at the m/z of 241.46 and (B) peak 3 at the m/z of 253.59.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5 739

BBREVE+DAIDZEIN_-VE #1297 RT: 24.80 AV: 1 NL: 1.17E6T: - c ESI Full ms [100.00-1000.00]

255.0 255.5 256.0 256.5 257.0 257.5 258.0 258.5 259.0 259.5 260.0m/z

0

100000

200000

300000

400000

500000

600000

700000

800000

900000

1000000

1100000

Inte

nsity

255.55

259.56

258.37257.40256.15

BBREVE+DAIDZEIN_-VE#1270 RT: 24.38 AV: 1 NL: 5.19E5T: - c ESI Full ms [100.00-1000.00]

250.0 250.5 251.0 251.5 252.0 252.5 253.0 253.5 254.0 254.5 255.0m/z

20253035404550556065707580859095

100 253.75

254.97

250.04

251.86

Rel

ativ

e Ab

unda

nce

1 2

3

4

1

2 3 4

5

A

B

Fig. 3 – Representative LC MS chromatograms of metabolites formed by the incubation of Bifidobacterium breve ATCC 15700

with daidzein using negative acquisition mode. (A) Peak 3 daidzein at the m/z of 253.75, (B) peak 1dihydrodaidzein at the m/z

of 255.55, (C) peak 8, 3-OH equol at the m/z of 288.11, (D) peak 1 equol at m/z of 241.32.

740 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5

fermentation times for the bacterial species were tested by one-

way ANOVA. Data were expressed as mean ± standard deviation

(STD). The statistical significance was considered at p < 0.05.

4. Results and discussion

4.1. Bioconversion of daidzein and equol production

While daidzein was anaerobically incubated in BHI broth with

a single culture of two Bifidobacterium strains (B. breve ATCC

15700 and B. longum BB536), equol was detected at 6 h following

the addition of daidzein to the medium (Fig. 1). The ability of

Bifidobacterium strains to transform daidzein to equol was

greatly increased over time (Table 1). Concentration of daidz-

ein changed significantly at the end of the incubation period.

The amount of equol produced by B. longum BB536 was slightly

higher compared to that of B. breve ATCC 15700 (Table 1).

It was found that Bifidobacterium strains efficiently metab-

olised daidzein to equol for the period of incubation. These

results differ from the findings of Raimondi et al. (2009)

BBREVE+DAIDZEIN_-VE #1423 RT: 26.88 AV: 1 NL: 5.28E5T: - c ESI Full ms [100.00-1000.00]

250 251 252 253 254 255 256 257 258 259 260m/z

0

10

20

30

40

50

60

70

80

90

100R

elat

ive

Abun

danc

e258.11

259.26252.18255.11

252.79256.61

251.34

250.40 255.76

1

2

3

4 5

6

7

8

9

BBREVE+DAIDZEIN_-VE #1912 RT: 35.68 AV: 1 NL: 3.75E5T: - c ESI Full ms [100.00-1000.00]

240.0 240.5 241.0 241.5 242.0 242.5 243.0 243.5 244.0 244.5 245.0m/z

0

50000

100000

150000

200000

250000

300000

350000

Inte

nsity

241.32

243.92242.02

1

23

c

d

Fig 3. (continued)

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5 741

Fig. 4 – Effect of daidzein on the growth of Bifidobacterium

breve 15700 .The growth curves for Bifidobacterium breve

15700 during incubation with and without daidzein were

compared and was found that there no significant different.

The values are the means of three experiments ± SD.

Fig. 5 – Effect of daidzein on the growth of Bifidobacterium

longum BB536. The growth curves for Bifidobacterium longum

BB536 during incubation with and without daidzein were

compared and was found that there no significant different.

The values are the means of three experiments ± SD.

Fig. 6 – Effect of daidzein on the pH of the fermentation

medium. The pH curve for BHI medium supplemented with

daidzein fermented with Bifidobacterium breve 15700 during

incubation with and without daidzein. The values are the

means of three experiments ± SD.

742 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5

However, these results agree with that of Tsangalis, Ashton,

McGill, & Shah, 2002, suggesting that Bifidobacterium animalis,

Bifidobacterium longum, and Bifidobacterium pseudolongum

hydrolysed soymilk isoflavone malonyl-, acetyl- and b-gluco-

sides to aglycones and transformed daidzein to equol. It was

also found that bifidobacteria transform equol from daidzein

(Tsangalis et al., 2002). As the genus Bifidobacterium is estab-

lished as b-glucosidase producer (Setchell et al., 2002; Tsang-

alis et al., 2002) the transformation of isoflavones glucosides

to aglycones could increase due to the presence of Bifidobacte-

rium strains during fermentation. However, a study using Lac-

tobacillus paracasei and Bifidobacterium longum co-cultures

found that the hydrolysis of isoflavones glucosides into agly-

cones increased from 36% to 90% of total isoflavones in soy-

milk (Chen, Su, & Wei, 2009). Other types of bifidobacteria

have effects on metabolic activity of the faecal equol produc-

tion in mice and humans. It was found that in faecal suspen-

sions from a human male equol producer, the equol

concentrations were lower in the control faecal suspension

than in the faecal suspension supplemented with a high con-

centration of strain TM-20 (Bifidobacterium isolated from the

human intestine) (Tamura, Saitoh, Tsushida, & Shinohara,

2006).

Twenty two Bifidobacterium strains were screened to inves-

tigate their ability to transform the isoflavones daidzin and

daidzein. These Bifidobacterium strains failed to transform

daidzein to equol, but most of the strains produced daidzein

from daidzin (Raimondi et al., 2009). Based on the diversity

among Bifidobacterium species and among strains of the same

species (Dabek, Mc-Crae, Stevens, Duncan, & Louis, 2008; Mar-

otti, Bonetti, Biavati, Catizone, & Dinelli, 2007; Tsangalis et al.,

2002), the production of equol from daidzein can be a strain-

dependent process. The metabolism of the isoflavone daidz-

ein by the intestinal bacteria is an important and valuable

matter to the bioavailability and bioactivity of this phytoes-

trogen (Rowland et al., 2003). Hence, bifidobacteria could be

used to improve the bioavailability and biotransformation of

isoflavones to equol. Equol has been reported to have greater

estrogenic activity and antioxidant capacity than its soy isofl-

avonoid precursor daidzein (Atkinson, Frankenfeld, & Lampe,

2005).

However, only 30–50% of humans have intestinal micro-

flora capable of converting daidzein to equol (Rufer, Glatt, &

Kulling, 2006). This fact has been suggested as an explanation

for the variance of results in dietary interference studies with

humans (Setchell et al., 2002). Our goals were to evaluate pro-

duction of equol by bifidobacteria and to investigate acid for-

mation in the medium used for incubation of bacteria in the

production of equol.

To confirm the correct identity of the results obtained by

HPLC, equol and daidzein standards and samples from incu-

bation medium of Bifidobacterium strains with daidzein were

analysed by LC–MS. The chromatograms obtained for the

standards are shown in Fig. 2A and B and for the samples

are shown in Fig. 3A–D.

4.2. Growth of Bifidobacterium strains

For both Bifidobacterium strains, the growth curves obtained

for the medium supplemented with daidzein were similar to

Fig. 7 – Effect of daidzein on the pH of the fermentation

medium. The pH curve for Bifidobacterium longum BB536

during incubation with and without daidzein. The values

are the means of three experiments ± SD.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5 743

those obtained in the absence of daidzein, thus indicating

that the presence of daidzein in the medium has no effect

on their growth (Figs. 4 and 5). Our results are in agreement

with those repoting that the 22 strains of Bifidobacterium have

the ability to grow in a medium containing daidzin and daidz-

ein (Raimondi et al., 2009).

4.3. pH of the medium during fermentation

During the incubation period of both Bifidobacterium strains,

the pH of the fermentation medium containing daidzein

was lower than that without daidzein (Figs. 6 and 7). These

findings can be explained by the fact that during the conver-

sion of daidzein to equol, short chain fatty acids such as lac-

tic, butyric, propionic and acetic acids are also released into

the fermentation medium, thus decreasing the pH (Setchell

et al., 2002). These results could be supported by the theory

that the production of acetic and lactic acids by bifidobacteria

is in mole ratio of 1.5 if pyruvate is converted to lactate (Des-

jardins, Roy, & Goulet, 1990). The present findings are parallel

to the study reporting that the concentration of short chain

fatty acid increased 30% during the supplementation period

due to significant (p < 0.05) increase in the formation of acetic

and propionic acids (De Boever, Deplancke, & Verstraete,

2000).

Table 2 – Formation of lactic acid during daidzein transformat

Time (h) Change

Bacteria with Daidzein

B. Breve B. longum

0 nd nd

6 2.14 ± 0.13a 4.04 ± 0.05

12 4.50 ± 0.05b 4.22 ± 0.03

18 4.89 ± 0.04c 4.36 ± 0.06

24 5.04 ± 0.05d 4.62 ± 0.02

48 5.20 ± 0.03d 4.83 ± 0.03

72 5.34 ± 0.04e 6.36 ± 0.08

96 5.53 ± 0.03f 6.83 ± 0.03

Values are means ± STD for triplicate independent experiments. Differen

differences between means. nd = Not detected-not determined.

4.4. Concentration of organic acids

The means of concentration (mmol l�1) of organic acids are

shown in Tables 2 and 3. The present study found that the

amount of acidification of the medium depended on the pres-

ence of daidzein and on the strains used for the fermentation.

This is in agreement with the study explaining, the acid resis-

tance behaviour of bifidobacteria was strain dependent (Matto

et al., 2004).

The concentrations of organic acids in the daidzein-sup-

plemented medium are higher compared to those without

daidzein. The concentration of lactic acid in the medium sup-

plemented with daidzein rapidly increased (p < 0.05) after 6 h

of incubation with B. breve ATCC 15700 (the concentration

ranged from 0 to 2.14 mmol l�1) and was high (the concentra-

tion was 5.53 mmol l�1 after 96 h) compared to lactic acid con-

centration in the medium without daidzein (the

concentration was 3.63 mmol l�1 after 96 h). On the other

hand, the concentration of lactic acid of B. longum BB536 in-

creased (p < 0.05) with fermentation time. The concentrations

ranged from 2.89 to 5.05 mmol l�1 and from 4.04 to

6.83 mmol l�1 for the medium without daidzein and the med-

ium supplemented with daidzein, respectively, after 96 h of

incubation. However, the amount of lactic acid produced by

B. longum BB536 was higher compared to that of B. breve ATCC

15700.

For the concentrations of acetic acid and lactic acid for B.

breve ATCC 15700 and B. longum BB536, they increased with

time. The concentration of acetic acid in the medium supple-

mented with daidzein and incubated with B. breve ATCC 15700

increased (p < 0.05) rapidly after 6 h of incubation (the con-

centration ranged from 0 to 5.89 mmol l�1) and was also high-

er (the concentration was 8.83 mmol l�1 after 96 h) compared

to the concentration of acetic acid in the medium not supple-

mented with daidzein (the concentration was 7.20 mmol l�1

after 96 h). In the case of incubation of B. longum BB536 in

medium not supplemented with daidzein, the concentration

of lactic acid increased (4.89–7.62 mmol l�1) while for the

medium supplemented with daidzein, the concentration ran-

ged from 5.42 to 8.75 mmol l�1 after 96 h of incubation. These

results show that the ratio of acetic acid concentration to lac-

tic acid was approximately 3 to 2, which is similar to the

ion (mmol l�1).

s in lactic acid concentrations

Bacteria without daidzein

B. Breve B. longum

nd nda 1.99 ± 0.05a 2.89 ± 0.03a

b 2.29 ± 0.04b 3.17 ± 0.16b

c 2.70 ± 0.04c 3.83 ± 0.03c

d 2.93 ± 0.03d 4.41 ± 0.02d

e 3.02 ± 0.03e 4.51 ± 0.02d

f 3.32 ± 0.02f 4.63 ± 0.01d

g 3.63 ± 0.03g 5.05 ± 0.06f

t superscript letters in the same column indicate significant (p < 0.05)

Table 3 – Formation of acetic acid during daidzein transformation (mmol l�1).

Time (h) Changes in acetic acid concentrations

Bacteria with Daidzein Bacteria without daidzein

B. Breve B. longum B. Breve B. longum

0 nd nd nd nd

6 5.89 ± 0.03a 5.42 ± 0.02a 2.50 ± 0.06a 4.89 ± 0.03a

12 7.07 ± 0.06b 6.32 ± 0.03b 3.51 ± 0.02b 5.72 ± 0.02b

18 7.51 ± 0.03c 7.09 ± 0.11c 5.08 ± 0.08c 5.92 ± 0.02c

24 7.82 ± 0.02d 8.13 ± 0.03d 5.46 ± 0.04d 6.49 ± 0.04d

48 8.59 ± 0.02e 8.58 ± 0.04e 6.06 ± 0.06e 6.60 ± 0.01e

72 8.71 ± 0.02f 8.66 ± 0.01e 6.80 ± 0.01f 7.51 ± 0.01f

96 8.83 ± 0.02g 8.75 ± 0.02e 7.20 ± 0.12g 7.62 ± 0.03g

Values are means ± STD for triplicate independent experiments. Different superscript letters in the same column indicate significant (p < 0.05)

differences between means. nd = Not detected-not determined.

744 J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5

results reported by others (Bezkorovainy & Miller-Catchpole,

1989; Scardovi & Trovatelli,1965). Usually, in a synthetic med-

ium, 3 moles of acetic acid and 2 moles of lactic acid are pro-

duced from 2 moles of glucose.

5. Conclusion

Both of the Bifidobacterium species studied were able to trans-

form daidzein to equol within 6 h of fermentation in a BHI

medium. The levels of equol increased during the incubation

period, indicating that equol was stable in the medium and

was not converted to other metabolites. The presence of

daidzein in the growth medium did not inhibit the growth of

Bifidobacterium strains. Excessive production of acetic and lac-

tic acids was noted in the medium supplemented with daidz-

ein for both of B. breve ATCC 15700 and B. longum (BB536),

which could be explained by the decrease in the pH of the fer-

mentation medium containing daidzein. Theoretically, bifido-

bacteria produce acetic and lactic acids in mole ratio of 1.5 if

pyruvate is converted to lactate. In this study, the Bifidobacte-

rium spp. strains were considered to have the possibility of

being used as functional food ingredients together with daidz-

ein or daidzein derivatives. Probiotic bacteria are widely used

to prepare fermented dairy products such as yoghurt or

freeze-dried cultures. Therefore, it is possible to use bifidobac-

teria and daidzein as food ingredients and supplements. In

addition to various health-related effects associated with the

intake of probiotics such as alleviation of lactose intolerance

and immune enhancement, some indications suggest the

roles of isoflavones (daidzein and equol) in reducing the risk

of breast and prostate cancers and some hormone-related dis-

eases. Therefore, attention should be paid to the microbiolog-

ical processes concerning biotransformation in order to find

ways to include this property into the diet.

R E F E R E N C E S

Adlercreutz, H. (2002). Phytoestrogens and cancer. Lancet Oncology,3, 364–373.

Akaza, H., Miyanaga, N., Takashima, N., Naito, S., Hirao, Y., &Tsukamoto, T. (2004). Comparisons of percent equol producers

between prostate cancer patients and controls: Case-controlled studies of isoflavones in Japanese, Korean andAmerican residents. Japanese Journal of Clinical Oncology, 34,86–89.

Atkinson, C., Berman, S., Humbert, O., & Lampe, J. (2004). In vitroincubation of human feces with daidzein and antibioticssuggests interindividual differences in the bacteriaresponsible for equol production. Journal of Nutrition, 134(3),596–599.

Atkinson, C., Frankenfeld, C. L., & Lampe, J. W. (2005). Gutbacterial metabolism of the soy isoflavone daidzein: Exploringthe relevance to human health. Journal of Experimental Biologyand Medicine, 230, 155–170.

Bezkorovainy, A., Miller-Catchpole, R. (1989). Biochemistry andphysiology of bifidobacteria. CRC.

Campbell, J. M., Fahey, G. C., & Wolf, B. W. (1997). Selectedindigestible oligosaccharides affect large bowel mass, cecaland fecal short-chain fatty acids, pH and microflora in rats.Journal of Nutrition, 127, 130–136.

Chen, T. R., Su, Q. R., & Wei, K. Q. (2009). Hydrolysis of isoflavonephytoestrogens in soymilk fermented by lactobacillus andbifidobacteria cocultures. Journal of Food Biochemistry, 34, 1–12.

Cummings, J., & Macfarlane, G. (1997). Role of intestinal bacteriain nutrient metabolism. Clinical Nutrition, 16, 3–11.

Dabek, M., Mc-Crae, S. I., Stevens, V. J., Duncan, S. H., & Louis, P.(2008). Distribution of b -glucosidase and b-glucuronidaseactivity and of b-glucuronidase gene gus in human colonicbacteria. FEMS Microbiology Ecology, 66, 487–495.

De Boever, P., Deplancke, B., & Verstraete, W. (2000). Fermentationby gut microbiota cultured in a simulator of the humanintestinal microbial ecosystem is improved by supplementinga soygerm powder. The Journal of Nutrition, 130, 2599–2606.

Decroos, K., Vanhemmens, S., Cattoir, S., Boon, N., & Verstraete,W. (2005). Isolation and characterization of an equol-producing mixed microbial culture from a human faecalsample and its activity under gastrointestinal conditions.Archives of Microbiology, 183, 45–55.

Desjardins, M. L., Roy, D., & Goulet, J. (1990). Growth ofbifidobacteria and their enzymes profiles. Journal of DairyScience, 73, 299–307.

Duncan, A., Phipps, W., & Kurzer, M. (2003). Phytoestrogens. Bestpractice and research. Journal of Clinical Endocrinology andMetabolism, 17, 253–271.

Gibson, G. R. (1998). Dietary modulation of the human gutmicroflora using prebiotics. British Journal of Nutrition, 80,S209–S212.

Gibson, G., McCartney, A., & Rastall, R. (2005). Prebiotics andresistance to gastrointestinal infections. British Journal ofNutrition, 93, 31–34.

J O U R N A L O F F U N C T I O N A L F O O D S 4 ( 2 0 1 2 ) 7 3 6 – 7 4 5 745

Hentges, D. J. (1980). Does diet influence human fecal microfloracomposition? Nutrition Reviews, 38, 329–336.

Hur, H., Beger, R., Heinze, T., Lay, J., Freeman, J., & Dore, J. (2002).Isolation of an anaerobic intestinal bacterium capable ofcleaving the C-ring of the isoflavonoid daidzein. Archives ofMicrobiology, 178, 8–12.

Kelly, G., Nelson, C., Waring, M., Joannou, G., & Reeder, A. (1993).Metabolites of dietary (soya) isoflavones in human urine.Clinica Chimica Acta, 223, 9–22.

Liong, M., & Shah, N. (2006). Effects of a Lactobacillus caseisynbiotic on serum lipoprotein, intestinal microflora, andorganic acids in rats. Journal of Dairy Science, 89, 1390–1399.

Lund, T., Munson, D., Haldy, M., Setchell, K., Lephart, E., & Handa,R. (2004). Equol is a novel anti-androgen that inhibits prostategrowth and hormone feedback. Biology of Reproduction, 70,1188–1195.

Marotti, I., Bonetti, A., Biavati, B., Catizone, P., & Dinelli, G. (2007).Biotransformation of common bean (Phaseolus vulgaris L.)flavonoid glycosides by Bifidobacterium species from humanintestinal origin. Journal of Agricultural and Food Chemistry, 55,3913–3919.

Maruo, T., Sakamoto, M., Ito, C., Toda, T., & Benno, Y. (2008).Adlercreutzia equolifaciens gen. nov., sp. nov., an equol-producing bacterium isolated from human faeces, andemended description of the genus Eggerthella. InternationalJournal of Systematic and Evolutionary Microbiology, 58, 1221–1227.

Matto, J., Malinen, E., Suihko, M. L., Alander, M., Palva, A., &Saarela, M. (2004). Genetic heterogeneity and functionalproperties of intestinal bifidobacteria. Journal of AppliedMicrobiology, 97, 459–470.

O’Sullivan, G. (2001). Probiotics. British Journal of Surgery, 88,161–162.

Parodi, P. W. (1999). The role of intestinal bacteria in the causationand prevention of cancer: Modulation by diet and probiotics.Australian Journal of Dairy Technology, 54, 103–121.

Rafii, F., Davis, C., Park, M., Heinze, T. M., & Beger, R. D. (2003).Variations in metabolism of the soy isoflavonoid daidzein byhuman intestinal microfloras from different individuals.Archives of Microbiology, 180, 11–16.

Raimondi, S. R. L., De Lucia, M., Amaretti, A., Leonardi, A., &Pagnoni, U. M. (2009). Bioconversion of soy isoflavones daidzinand daidzein by Bifidobacterium strains. Applied Microbiologyand Biotechnology, 81, 943–950.

Reinli, K., & Block, G. (1996). Phytoestrogens content of foods acompendium of literature values. Nutrition and Cancer, 26,123–148.

Rowland, I., Faughnan, M., Hoey, L., Wahala, K., Williamson, G., &Cassidy, A. (2003). Bioavailability of phytoestrogens. The BritishJournal of Nutrition, 89, S45.

Rufer, C. E., Glatt, H., & Kulling, S. E. (2006). Structural elucidationof hydroxylated metabolites of the isoflavan equol by gaschromatography-mass spectrometry and high-performanceliquid chromatography mass spectrometry. Drug Metabolismand Disposition, 34, 51–60.

Sathyamoorthy, N., & Wang, T. (1997). Differential effects ofdietary phytoestrogens daidzein and equol on human breastcancer MCF-7 cells. European Journal of Cancer, 33, 2384–2389.

Scardovi, V., & Trovatelli, L. D. (1965). The fructose-6-phosphateshunt as a peculiar pattern of hexose degradation in the genus

Bifidobacterium. Annual of Microbiology and Enzymology, 15,19–27.

Setchell, K., & Adlercreutz, H. (1988). Mammalian lignans andphytoestrogens: Recent studies on their formation,metabolism and biological role in health and disease. Role ofthe Gut Flora in Toxicity and Cancer, 1988, 315–345.

Setchell, K., Borriello, S., Hulme, P., Kirk, D., & Axelson, M. (1984).Nonsteroidal estrogens of dietary origin: Possible roles inhormone-dependent disease. American Journal of ClinicalNutrition, 40, 569.

Setchell, K., Brown, N., Zimmer-Nechemias, L., Brashear, W.,Wolfe, B., & Kirschner, A. (2002). Evidence for lack ofabsorption of soy isoflavone glycosides in humans, supportingthe crucial role of intestinal metabolism for bioavailability.American Journal of Clinical Nutrition, 76, 447–453.

Setchell, K. D. R., & Cassidy, A. (1999). Dietary isoflavones:Biological effects and relevance to human health. Journal ofNutrition, 129, 758S–767S.

Setchell, K., Lawson, A., Mitchell, F., Adlercreutz, H., Kirk, D., &Axelson, M. (1980). Lignans in man and in animal species.Nature, 287, 740–742.

Setchell, K., Zimmer-Nechemias, L., Cai, J., & Heubi, J. (1998).Isoflavone content of infant formulas and the metabolic fateof these phytoestrogens in early life. American Journal of ClinicalNutrition, 68, 1453S–1461S.

Shimada, Y., Yasuda, S., Takahashi, M., Hayashi, T., Miyazawa, N.,& Sato, I. (2010). Cloning and expression of a novel NADP (H)-dependent daidzein reductase, an enzyme involved in themetabolism of daidzein, from equol-producing lactococcusstrain 20–92. Applied of Environmental Microbiology, 76,5892–5901.

Tamura, D., Saitoh, H., Tsushida, T., & Shinohara, K. (2006).Bifidobacterium may affect in vitro metabolism of daidzein byfecal flora from mice and a human male equol producer.Microbial Ecology in Health and Disease, 18, 42–46.

Thawornkuno, C., Tanaka, M., Sone, T., & Asano, K. (2009).Biotransformation of daidzein to equol by crude enzyme fromAsaccharobacter celatus AHU1763 required an anaerobicenvironment. Bioscience, Biotechnology and Biochemistry, 73,1435–1438.

Tsangalis, D., Ashton, J., McGill, A., & Shah, N. (2002). Enzymictransformation of isoflavone phytoestrogens in soymilk byglucosidase producing bifidobacteria. Journal of Food Science, 67,3104–3113.

Turner, N. J., Thomson, B. M., & Shaw, I. C. (2003). Bioactiveisoflavones in functional foods: The importance of gutmicroflora on bioavailability. Nutrition Reviews, 61, 204–213.

Wang, X., Hur, H. G., Lee, J. H., Kim, K. T., & Kim, S. (2005).Enantioselective synthesis of S-equol from dihydrodaidzein bya newly isolated anaerobic human intestinal bacterium.Applied and Environmental Microbiology, 71, 214–219.

Wang, X. L., Kim, J. H., Kang, S., Kim, S., & Hur, G. H. (2007).Production of phytoestrogens S-equol from daidzein in mixedculture of two anaerobic bacteria. Archives of Microbiology, 187,155–160.

Wang, X. L., Shin, K. H., Hur, H. G., & Kim, S. (2005). Enhancedbiosynthesis of dihydrodaidzein and dihydrogenistein by anewly isolated bovine rumen anaerobic bacterium. Journal ofBiotechnology, 115, 261–269.