www.elsevier.com/locate/ijfoodmicro

International Journal of Food Microbiology 94 (2004) 79–86

Effect of the adaptation to high bile salts concentrations on

glycosidic activity, survival at low PH and cross-resistance to

bile salts in Bifidobacterium

Luis Noriega, Miguel Gueimonde, Borja Sanchez,Abelardo Margolles, Clara G. de los Reyes-Gavilan*

Instituto de Productos Lacteos de Asturias, CSIC, Ctra. de Infiesto s/n. 33300 Villaviciosa, Asturias, Spain

Received 17 February 2003; received in revised form 23 July 2003; accepted 2 January 2004

Abstract

Six derivatives with increased resistance to ox gall (MIC: z 1% w/v) and one derivative resistant to sodium cholate (MIC:

0.8% w/v) were obtained from more sensitive original Bifidobacterium strains. These microorganisms, and two additional

cholate resistant derivatives obtained in a previous study (Int. J. Food Microbiol. 82 (2003) 191), were partially characterised in

this study. Acquisition of resistance against a given bile salt, also conferred cross-resistance to other bile salts, and promoted an

increase in the survival of these microorganisms at low pH. Bile resistance levels of derivatives were dependent on the external

pH so that the resistance was lower at neutral pH values than in acidic environments. In addition, the acquisition of bile

resistance induced changes on glycoside-hydrolysing activities of derivatives obtained from five out of eight original strains,

with certain activities such as h-glucosidase showing more than tenfold increases in some of these microorganisms. These data

suggest that the exposure to high bile salts concentrations may have induced a synergic response on Bifidobacterium for the

adaptation to the conditions of the gastrointestinal tract. This could have improved the survival at low pH in these

microorganisms, the resistance to high bile salts concentrations, and the assimilation of non-digestible carbohydrates by the

enhancement of some glycoside-hydrolysing activities.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Bifidobacterium; Bile resistance; Cholate; Ox gall; Glycosidic activity; Low pH

1. Introduction

Bifidobacteria are considered as probiotics, and

are being used as active ingredients in functional

dairy-based products. The health-promoting effects

0168-1605/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.ijfoodmicro.2004.01.003

* Corresponding author. Tel.: +34-985-892131; fax: +34-985-

892233.

E-mail address: greyes_gavilan@ipla.csic.es

(C.G. de los Reyes-Gavilan).

attributed to these microorganisms, include the inhi-

bition of pathogens, antimutagenic and anticarcino-

genic activity, prevention of diarrhoea, stimulation of

the immune response and a reduction of serum

cholesterol levels (Tannock, 1999). Some Bifidobac-

terium strains can colonise the gastrointestinal tract

(GIT), and are important components of the human

intestinal microbiota, in which they occur at concen-

trations of 109–1010 cells/g of faeces (Tannock,

1995). To achieve this colonisation, these bacteria

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–8680

must overcome biological barriers that include acid

in the stomach and bile in the intestine (Gilliland,

1978; Kanbe, 1992; Lankaputhra and Shah, 1995).

During digestion, the time from entrance to release

of food from the stomach is around 90 min (Berrada

et al., 1991) at a pH as low as 1.5 (Lankaputhra and

Shah, 1995) or 2.0 (Hill, 1990). Further digestive

processes have longer residence times. Davenport

(1977) reported that bile concentration ranged from

1.5% to 2% w/v in the first hour of digestion, and

the levels decreased afterwards to around 0.3% w/v

(Sjovall, 1959; Gilliland et al., 1984). Bile salts are

synthesised in the liver from cholesterol, secreted as

conjugates of either glycine or taurine into the

duodenum, where they facilitate fat absorption and

undergo enterohepatic circulation (Hofmann, 1984).

During the enterohepatic circulation, bile salts can

undergo two major modifications by the intestinal

microflora. Deconjugation of bile salts by bile salt

hydrolases, results in the formation of primary bile

acids (cholic and quenodeoxycholic), which may be

subsequently 7a-dehydroxylated into secondary bile

acids (deoxycholic and lithocholic) (Baron and Hyle-

mon, 1997). Bile acids are toxic for living cells.

Therefore, the autochthonous gastrointestinal micro-

biota must have developed strategies to defend

themselves against the toxic action of these com-

pounds. Although the resistance mechanisms of these

bacteria are still poorly understood, the inhibition of

Bifidobacterium growth by bile salts may be over-

come in some cases by progressive adaptation to

increasing concentrations of these compounds (Grill

et al., 1995; Chung et al., 1999; Margolles et al.,

2003).

Some of the main substrates for bacterial growth in

the colon are dietary carbohydrates that were not

digested in the upper GIT. These carbohydrates are

assimilated preferentially by bifidobacteria, promot-

ing their proliferation in the gut. The ability of these

microorganisms to metabolise non-digestible carbo-

hydrates is dependent on their glycosidic activities.

On the other hand, De Smet et al. (1995) suggested

that a metabolisable carbon source could alleviate the

toxic effect of bile salts in lactobacilli, and Perrin et

al. (2000) demonstrated that the resistance to bile salts

of some Bifidobacterium species increased in the

presence of fructooligosaccharides. A study by Row-

land and Tanaka (1993), showed that h-glucosidase

activity of caecal bacteria was increased in rats

colonised with a human faecal microflora and fed

with transgalactosylated oligosaccharides. However,

other reports related microbial h-glycosidase activi-

ties with the level of mutagenic or carcinogenic

aglycones from plant glycosides in the gut (Rowland

et al., 1985; Goldin, 1986).

In a recent study we described changes of mem-

brane proteins, morphology and carbohydrate fer-

mentation profiles of a Bifidobacterium strain with

acquired resistance to cholate (Margolles et al.,

2003). In this context, the aim of the present study

was to elucidate whether a relation exists between the

resistance to bile salts, and two important character-

istics of bifidobacteria, for their survival and coloni-

sation of the intestine: the tolerance to low pH and

glycosidic activities.

2. Material and methods

2.1. Bacterial strains

Bifidobacterium strains were cultured under anaer-

obic conditions (Anaerocult A System; Merck, Darm-

stadt, Germany) for 24–48 h at 37 jC in MRS broth

(Merck) supplemented with 0.05% w/v L-cysteine

(Merck) (MRSC).

2.2. Sensitivity of Bifidobacterium strains to bile salts

and isolation of bile salts-resistant derivatives

Twenty-nine strains were initially used in an

attempt to obtain derivatives resistant to higher ox

gall or sodium cholate concentrations. For this pur-

pose incubations of 1% v/v inocula were carried out

for 2–7 days in MRSC broth, supplemented with

gradually increasing concentrations of sodium cholate

and ox gall, i.e. 0.05%, 0.1%, 0.2%, 0.3%, 0.4%,

0.5%, 0.6%, 0.7%, 0.8%, 0.9% and 1.0% (w/v). Six

derivatives with increased resistance to ox gall (dOx)

and one derivative resistant to high cholate (dCo)

concentrations, were obtained from the more sensi-

tive strains of origin (Table 1). In addition, two

cholate resistant derivatives obtained in a previous

work, 4549dCo and M6dCo (Margolles et al., 2003),

and their respective original strains, were included in

this study.

Table 1

Susceptibility in MRS agar supplemented with 0.05% w/v L-cysteine (MRSC) and in buffered-MRSC agar to sodium cholate, ox gall and

sodium deoxycholate (MIC, % w/v) of Bifidobacterium strains and their resistant derivatives originally obtained against ox gall (dOx) or sodium

cholate (dCo)

Strainsa Source Cholate Ox gall Deoxycholate

MRSC Buffered-

MRSC

MRSC Buffered-

MRSC

MRSC Buffered-

MRSC

B. longum NIZO B667 – 0.20 0.20 0.25 0.25 0.10 0.10B. longum B667dCo 0.80 0.20 1.00 0.50 0.20 0.20B. breve NIZO B658 Infant faeces 0.20 0.20 0.50 0.50 0.20 0.20B. breve B658dOx 0.80 0.40 2.00 0.50 0.40 0.20B. bifidum CECT4549 Nursling stools 0.10 0.10 0.12 0.12 0.10 0.10B. bifidum 4549dCo 0.80 0.20 2.00 0.25 0.40 0.20B. bifidum 4549dOx 0.80 0.20 2.00 0.25 0.40 0.20B. infantis ATCC 15697 Infant intestine 0.20 0.20 0.50 0.50 0.20 0.20B. infantis 15697dOx 0.80 0.40 2.00 0.50 0.40 0.20B. bifidum PBT Probiotic supplement 0.20 0.20 0.25 0.25 0.20 0.20B. bifidum PBTdOx 0.80 0.40 >2.00 1.00 0.40 0.20B. bifidum A1 Commercial dairy culture 0.20 0.20 0.25 0.25 0.20 0.20B. bifidum A1dOx 0.80 0.40 2.00 0.50 0.40 0.20B. bifidum A8 Commercial 0.40 0.40 0.50 0.50 0.20 0.20B. bifidum A8dOx dairy culture 0.80 0.40 1.00 0.50 0.40 0.20B. bifidum M6 Commercial 0.20 0.20 0.50 0.50 0.20 0.20B. bifidum M6dCo fermented milk 0.80 0.40 >2.00 1.00 0.40 0.20

a ATCC: American Type Culture Collection, Rockville, USA. CECT: Spanish Collection of Type Cultures, Valencia, Spain. NIZO: NIZO

Food Research Culture Collection, Ede, The Netherlands.

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–86 81

Minimum inhibitory concentration (MIC) determi-

nations of original strains and their corresponding bile-

resistant derivatives, were made on MRSC agar sup-

plemented with twofold serial dilutions of ox gall

(Sigma, St. Louis, MO, USA), from 2% to 0.062%

w/v, or sodium cholate, from 0.8% to 0.025% w/v

(Sigma). The plates were incubated for 48 h at 37 jCunder anaerobic conditions.

The possible acquisition of cross-resistance to

other bile salts, and the influence of the external

pH on bile resistance levels, were evaluated in the

resistant derivatives and original strains. For this

purpose, MICs were determined as described above

at twofold increasing concentrations of ox gall (from

0.062% to 2.0% w/v), sodium cholate (from 0.025%

to 0.8% w/v) and sodium deoxycholate (0.0125% to

0.4% w/v), both in MRSC agar and in MRSC agar

buffered with 0.08 M PIPES pH 6.4 (Sigma) (buff-

ered-MRSC agar).

Experiments were carried out in duplicate using

independent inocula. Variations between both meas-

ures did not exceed one dilution, either higher or

lower.

2.3. Tolerance to low pH

The tolerance against acid of Bifidobacterium

strains and their resistant derivatives was determined.

Amounts of 0.2 ml of cells grown overnight at 37 jCwere centrifuged, washed in NaCl 0.5% w/v and

resuspended in 2.0 ml of an acid solution (NaCl

0.5% w/v adjusted to pH 2.0 with HCl) obtaining

final counts between 107 and 108 cfu/ml. Aliquots of

0.1 ml were removed from acid solution after 90 min

of incubation at 37 jC and counted in MRSC agar,

(Merck) as described before. Results were expressed

as count decreases in cfu/ml with respect to the counts

in a control solution (NaCl 0.5% w/v) after incuba-

tion. Experiments were made in duplicate.

2.4. Glycoside-hydrolysing activities

Glycosidase activities of original strains and bile-

resistant derivatives, were assayed on cell-free

extracts using different p-NP-glycoside substrates

(Sigma). Bifidobacterium cells grown anaerobically

at 37 jC in 20 ml MRSC to OD600 1.2 were harvested

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–8682

by centrifugation, washed with 100 mM potassium

phosphate buffer pH 6.8, and pellets were resus-

pended in 1 ml of the same buffer. Cells were

sonicated for 2 min while cooling on ice, using a

CV17 sonicator (VibraCell, Sonics and Materials,

Connecticut, USA) and centrifuged to remove cell

debris. Extracts obtained were then assayed for gly-

cosidase activities. Amounts of 40 Al of cell-free

extracts were mixed with 880 Al of 40 mM acetate

buffer, pH 5.5 and 80 Al of 20 mM of the

corresponding p-NP-glycoside. The mixtures were

incubated in a water-bath at 37jC for 10 min. After

incubation, 1 ml of cold 1 M sodium carbonate was

added to stop the reaction. The calculation of micro-

moles of p-nitrophenol (p-NP) released was based on

the relationship of the A420 nm to a standard curve.

One unit of enzyme activity was defined as the

amount that releases 1 Amol of p-NP per min. Protein

of cell-free extracts was determined using the BCA

protein assay kit (Pierce, Rockford, IL, USA) follow-

ing the manufacturer’s instructions. The activities

were given as specific activities (units/mg) in cell-free

extracts. Experiments were made in triplicate using

independent cultures each time.

Table 2

Susceptibility to acid (pH 2.0) of Bifidobacterium strains and their

resistant derivatives originally obtained against ox gall (dOx) or

sodium cholate (dCo)

Straina Count decreases (log cfu/ml)

in acid solution (pH 2.0) after

90 min exposure

B. longum NIZO B667 5.74B. longum B667dCo 5.54B. breve NIZO B658 2.30B. breve B658dOx 1.03B. bifidum CECT4549 4.79B. bifidum 4549dCo 1.63B. bifidum 4549dOx 2.13B. infantis ATCC 15697 6.04B. infantis 15697dOx 2.30B. bifidum PBT 2.97B. bifidum PBTdOx 2.23B. bifidum A1 3.79B. bifidum A1dOx 1.90B. bifidum A8 6.68B. bifidum A8dOx 0.61B. bifidum M6 2.63B. bifidum M6dCo 0.92

a ATCC: American Type Culture Collection, Rockville, USA.

CECT: Spanish Collection of Type Cultures, Valencia, Spain. NIZO:

NIZO Food Research Culture Collection, Ede, The Netherlands.

3. Results and discussion

Derivatives with either two to sixteen-fold in-

creased resistance to ox gall (MIC: z 1 % w/v) or

four- to eightfold increased resistance to sodium

cholate (MIC: 0.8% w/v), were obtained in this and

in a previous study (Margolles et al., 2003), from

more sensitive original Bifidobacterium strains from

different sources (Table 1). All resistant derivatives

displayed increased resistance to deoxycholate, cho-

late and ox gall, independently to the compound (ox

gall or cholate) initially used for selection. This

indicated that the acquisition of resistance against a

bile salt or a mixture of them also conferred cross-

resistance to other bile salts.

Adaptation to high concentrations of bile salts and

to low pH, might be valuable tools for increasing the

survival of bifidobacteria in the GIT. Therefore, we

looked for a possible relation between bile resistance

levels and tolerance to low pH that could confer an

additional selective advantage to these microorgan-

isms. For this purpose, the capacity to survive in acid

solution (pH 2.0) was tested against original strains

and resistant derivatives. As shown in Table 2, in

general the derivatives displayed considerably higher

survival at 90 min of exposure to low pH, than their

corresponding strains of origin. It seems that the

increase of resistance to ox gall or cholate induced

on Bifidobacterium could have also conferred on this

microorganism a greater capacity to tolerate the low

pH, probably making it better adapted to surviving

through the GIT. In addition, MICs against ox gall,

cholate and deoxycholate of our resistant derivatives

grown in buffered-MRSC medium, were considerably

lower than MICs obtained in unbuffered medium

(Table 1). However, no variations on MIC levels were

obtained for the original strains when the culture

mediumwas initially buffered. These results evidenced

that bile resistance of our derivatives, but not of the

corresponding original strains, was dependent on the

external pH being lower at neutral pH than in acidic

environments. These data pointed to a narrow relation-

ship between bile resistance and tolerance to low pH in

Bifidobacterium. Chung et al. (1999) and Prasad et al.

(1998) selected bile salt resistant strains of Bifidobac-

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–86 83

terium from human faeces that appeared to be also

resistant to low pH.

In order to know whether the acquisition of bile

salt-resistance may have induced changes on glyco-

sidic activities of resistant derivatives, activities to-

wards some p-NP-glycosides were quantified in cell-

free extracts obtained from cultures grown until early

stationary phase (Table 3). Activity against p-NP h-D-glucuronide, related to the conversion of precarcino-

gens to carcinogens, was not detected in any strain

(data not shown). Microorganisms tested generally

showed high activity levels towards p-NP a-D-galac-

topyranoside, p-NP h-D-galactopyranoside and p-NP

a-D-glucopyranoside, which was in agreement with

results previously obtained by other authors (Guei-

monde et al., unpublished results; Desjardins et al.,

1990). In addition, all of them presented activity

towards p-NP h-D-fucopyranoside. Arbitrarily, we

considered significant changes on glycosidic activity

levels of resistant derivatives when more than three-

fold increases or decreases, with respect to the activity

of the original strain, were measured. According to

this criterion, resistant derivatives obtained from five

out of the eight original strains considered in this

work, displayed changes on glycosidic activity. The

activity towards p-NP a-L-arabinofuranoside in-

creased in B. longum B667dCo and activities towards

p-NP N-acetyl-h-D-glucosaminide and p-NP a-L-

rhamnopyranoside increased in B. breve B658dOx,

although these enzymatic activities remained at low

levels in both microorganisms. The most noteworthy

changes were evidenced for resistants derived from B.

bifidum CECT 4549, B. infantis ATCC 15697 and B.

bifidum PBT. Thereby, B. bifidum 4549dOx and B.

infantis 15697dOx showed decreases on p-NP h-D-galactopyranoside, p-NP a-D-galactopyranoside and

p-NP N-acetyl-h-D-glucosaminide hydrolysing activi-

ties, whereas B. bifidum 4549dCo had lower activities

against p-NP a-D-galactopyranoside and p-NP N-ace-

tyl-h-D-glucosaminide. These results are interesting

because a-D-galactosidase and N-acetyl-h-D-glucosa-minidase have been related to the breakdown of

human glycoproteins (Oakey et al., 1995). The resis-

tant derivatives B. bifidum 4549dCo, B. bifidum

4549dOx and B. infantis 15697dOx also displayed

increased activities towards p-NP a-D-glucopyrano-

side, p-NP h-D-glucopyranoside and p-NP h-D-fuco-pyranoside. On the other hand, B. bifidum PBTdOx

showed lower hydrolysing activity towards p-NP a-L-

arabinofuranoside, and higher activity levels towards

p-NP h-D-galactopyranoside, p-NP h-D-fucopyrano-side and p-NP h-D-glucopyranoside. Tochiura et al.

(1986) suggested that the hydrolysis of h-D-fucosideon some bifidobacteria could be catalysed by h-D-galactosidases or h-D-glucosidases, and Schell et al.

(2002) recently pointed to the lack of sequences of

homologous enzymes needed for fucose fermentation

in the genome of B. longum. The simultaneous

increase of activities towards p-NP h-D-fucopyrano-side and p-NP h-D-glucopyranoside in our bile resis-

tant derivatives obtained from three strains (B. bifidum

CECT 4549, B. infantis ATCC 15697 and B. bifidum

PBT), supported the hypothesis of the hydrolysis of

fucose by enzymes other than specific fucosidases

such as h-glucosidases. It seems evident that the

acquisition of bile salt resistance induced significant

changes on glycoside-hydrolising activities, with lev-

els of certain activities decreasing, and others showing

more than ten-fold increases in some resistant deriv-

atives, with respect to their original strains. Relating

to that, Zarate et al. (2000) indicated that bile-tolerant

strains of propionibacteria showed higher synthesis of

h-galactosidase in the presence of bile salts, and

Perrin et al. (2000) demonstrated that the resistance

to bile salts of some Bifidobacterium species in-

creased in the presence of fructooligosaccharides.

It could be hypothesized that the exposure to high

bile salt concentrations may have induced a synergic

response on some Bifidobacterium strains for the

adaptation to the GIT conditions. This could have

improved for these microorganisms, the resistance to

some of the conditions of the GIT (low pH in the

stomach and high bile salt concentrations in the

intestine), and the assimilation of non-digestible car-

bohydrates in the gut. Marteau et al. (1990) indicated

that sequential gastric and intestinal passage produced

a synergic reduction on the survival of probiotics

through the GIT. In that way, changes found in bile

resistance levels of our derivatives as a function of the

external pH, might also reflect a physiological adap-

tation to pH variations through the GIT. The previous

exposure to low pH in the stomach, might cause a

transitory rise of bile resistance, increasing the sur-

vival of these microorganisms at high bile concentra-

tion in the duodenum, whereas the increase of pH in

the small bowel might promote a decrease of the

Table 3

Specific glycosidase activities (units/mg protein) of Bifidobacterium strains and their resistant derivatives obtained against ox gall (dOx) or sodium cholate (dCo). Arrows indicate more than threefold increases (z) or decreases (#) on the

activity of resistant derivatives with respect to the original strains

Strainsa Substrate

U-NP

N-Acetyl-h-D-glucosaminide

U-NP a-L-

Arabinofuranoside

U-NP h-D-Fucopyranoside

U-NP a-D-

Galactopyranoside

U-NP h-D-Galactopyranoside

U-NP a-D-

Glucopyranoside

U-NP h-D-Glucopyranoside

U-NP a-L-

Rhamnopyranoside

U-NP h-D-Xylopyranoside

B. longum NIZO B667 20.72 6.59 19.64 162.25 248.63 320.12 0.00 0.00 0.00

B. longum B667dCo 20.62 26.07z 14.92 180.31 237.81 181.93 0.00 0.00 0.00

B. breve NIZO B658 0.57 6.52 286.06 37.93 231.84 360.74 355.87 1.26 2.07

B. breve B658dOx 4.69 z 4.79 252.84 36.89 152.11 249.04 363.30 9.08 z 4.73

B. bifidum CECT4549 17.09 24.99 18.45 145.84 393.74 91.56 0.00 0.00 1.19

B. bifidum 4549dCo 0.00 # 13.00 238.95 z 41.52 # 144.81 393.74 z 275.53 z 0.00 2.76

B. bifidum 4549dOx 3.74 # 10.46 193.95 z 20.73 # 73.63 # 595.12 z 245.67 z 0.00 2.01

B. infantis ATCC 15697 227.44 36.44 55.39 191.39 3699.94 260.67 30.55 0.00 0.00

B. infantis 15697dOx 18.82 # 72.09 357.99 z 43.32 # 154.40 # 1236.15 z 371.31 z 0.00 0.00

B. bifidum PBT 0.00 139.18 27.58 94.75 19.21 353.34 38.30 0.00 0.00

B. bifidum PBTdOx 1.94 z 17.21 # 220.30 z 41.50 73.49 z 358.00 404.14 z 0.00 0.00

B. bifidum A1 8.39 52.06 194.03 25.43 209.06 260.15 235.60 0.00 2.84

B. bifidum A1dOx 3.82 54.07 195.90 16.56 133.12 159.34 235.73 0.00 3.13

B. bifidum A8 209.73 0.00 43.76 17.83 1651.32 20.00 0.00 0.00 0.00

B. bifidum A8dOx 212.14 0.00 52.44 22.44 966.81 23.15 0.00 0.00 0.00

B. bifidum M6 0.00 12.01 247.41 43.68 133.82 310.73 783.31 0.00 0.00

B. bifidum M6dCo 0.00 32.24 242.20 72.68 149.16 319.74 360.67 0.00 0.00

a ATCC: American Type Culture Collection, Rockville, USA. CECT: Spanish Collection of Type Cultures, Valencia, Spain. NIZO: NIZO Food Research Culture Collection, Ede, The Netherlands.

L.Norieg

aet

al./Intern

atio

nalJournalofFoodMicro

biology94(2004)79–86

84

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–86 85

resistance levels on Bifidobacterium derivatives, as

bile salt concentration decreased. Finally and related

to that, it is worth noting that Gomez-Zavaglia et al.

(2002) recently reported a change on surface proper-

ties of bifidobacteria as a response to long-term

exposure of bacteria to bile and indicated that bile

action could be related to irreversible metabolic

changes in these microorganisms.

The relation between bile salts resistance, survival

at low pH, and glycosidic activity patterns on Bifido-

bacterium, requires further studies.

Acknowledgements

This work was financed by European Union

FEDER funds and the Spanish Plan Nacional de

I +D (project AGL2001-2296). Luis Noriega and

Borja Sanchez were the recipients of a predoctoral

fellowship from the Fundacion para la Investigacion

Cientıfica y Tecnica (Asturias, Spain) (FICYT) and a

predoctoral FPI fellowship from the Spanish Minis-

terio de Ciencia y Tecnologıa, respectively. M.

Gueimonde was supported by a contract funded under

project AGL2001-2296. Strains B. bifidum PBT, A1

and A8 were kindly provided by Dr. J.A. Reinheimer,

Universidad Nacional del Litoral, Santa Fe, Argen-

tina. We thank NIZO Food Research for providing us

with the strains B667 and B658.

References

Baron, S.F., Hylemon, P.B., 1997. Biotransformation of bile acids,

cholesterol, and steroid hormones. In: Mackie, R.I., White, B.A.

(Eds.), Gastrointestinal Ecosystems and Fermentations. Gastro-

intestinal Microbiology, vol. I. International Thompson Publish-

ing, New York, pp. 470–510.

Berrada, N., Lemeland, J.F., Laroch, G., Thouvenot, P., Piaia, M.,

1991. Bifidobacterium from fermented milks: survival during

gastric transit. J. Dairy Sci. 74, 409–413.

Chung, H.S., Kim, Y.B., Chun, S.L., Ji, G.E., 1999. Screening and

selection of acid and bile resistant bifidobacteria. Int. J. Food

Microbiol. 47, 25–32.

Davenport, H.W., 1977. Physiology of the Digestive Tract, 4th

edition. Year Book Medical, Chicago, IL.

Desjardins, M.J., Roy, D., Goulet, J., 1990. Growth of bifidobac-

teria and their enzyme profiles. J. Dairy Sci. 73, 299–307.

De Smet, I., van Hoorde, L., van de Woestyne, M., Cristiaens, H.,

Verstraete, W., 1995. Significance of bile salt hydrolytic activ-

ities of lactobacilli. J. Appl. Bacteriol. 79, 292–301.

Gilliland, S.E., 1978. Beneficial inter-relationships between certain

micro-organisms and humans: candidate micro-organisms for

use as dietary adjuncts. J. Food Prot. 42, 164–167.

Gilliland, S.E., Staley, T.E., Bush, L.J., 1984. Importance of bile

tolerance of Lb. acidophilus used as a dietary adjunct. J. Dairy

Sci. 67, 3045–3051.

Goldin, B.R., 1986. The metabolism of the intestinal microflora and

its relationship to dietary fat, colon and breast cancer. Prog.

Clin. Biol. Res. 222, 655–685.

Gomez-Zavaglia, A., Kociubinski, G., Perez, P., Disalvo, E., De

Antoni, G., 2002. Effect of bile on the lipid composition and

surface properties of bifidobacteria. J. Appl. Microbiol. 93,

794–799.

Grill, J.P., Manginot-Durr, C., Schneider, F., Ballongue, J., 1995.

Bifidobacteria and probiotics effects: action of Bifidobacte-

rium species on conjugated bile salts. Curr. Microbiol. 31,

23–27.

Hill, M.J., 1990. Factors controlling the microflora of the

health upper gastrointestinal tract. In: Hill, M.J., Marsh,

P.D. (Eds.), Human Microbial Ecology. CRC Press, Boca

Raton, FL, pp. 57–85.

Hofmann, A.F., 1984. Chemistry and enterohepatic circulation of

bile acids. Hepatology 4, 4S–14S.

Kanbe, M., 1992. Cancer control and fermented milk. In: Naka-

zawa, Y., Hosono, A. (Eds.), Functions of Fermented Milk:

Challenges for Health Sciences. Elsevier, London, UK, p. 377.

Lankaputhra, W.E.V., Shah, N.P., 1995. Survival of Lactobacillus

acidophilus and Bifidobacterium ssp. in the presence of acid and

bile salts. Cult. Dairy Prod. J. 30, 2–7.

Margolles, A., Garcıa, L., Sanchez, B., Gueimonde, M., de los

Reyes-Gavilan, G.C., 2003. Characterisation of a Bifidobacte-

rium strain with acquired resistance to cholate-A preliminary

study. Int. J. Food Microbiol. 82, 191–198.

Marteau, P., Pochart, P., Flourie, B., Pellier, P., Santos, L., Desjeux,

J.F., Rambaud, J.C., 1990. Effect of chronic ingestion of fer-

mented dairy product containing Lactobacillus acidophilus and

Bifidobacterium bifidum on metabolic activities on the colonic

flora in humans. Am. J. Clin. Nutr. 52, 685–688.

Oakey, H.H., Harty, D.W.S., Knox, K.W., 1995. Enzyme produc-

tion by lactobacilli and the potential link with infective endo-

carditis. J. Appl. Bacteriol. 78, 142–148.

Perrin, S., Grill, J.P., Schneider, F., 2000. Effects of fructooligo-

saccharides and their monomeric components on bile salt re-

sistance in three species of bifidobacteria. J. Appl. Microbiol.

88, 968–974.

Prasad, J., Gill, H., Smart, J., Gopal, P.K., 1998. Selection and

characterisation of Lactobacillus and Bifidobacterium strains

for use as probiotics. Int. Dairy J. 8, 993–1002.

Rowland, I.R., Tanaka, R., 1993. The effects of transgalactosylated

oligosaccharides on gut flora metabolism in rats associated with

a human faecal microflora. J. Appl. Bacteriol. 74, 667–674.

Rowland, I.R., Mallett, A.K., Wise, A., 1985. The effect of diet on

the mammalian gut flora and its metabolic activities. CRC Crit.

Rev. Toxicol. 16, 31–103.

Schell, M.A., Karmirantzou, M., Snel, B., Vilanova, D., Berger, B.,

Pessi, G., Zwahlen, M.C., Desiere, F., Bork, P., Delley, M.,

Pridmore, R.D., Arigoni, F., 2002. The genome sequence of

L. Noriega et al. / International Journal of Food Microbiology 94 (2004) 79–8686

Bifidobacterium longum reflects its adaptation to the human

gastrointestinal tract. Proc. Natl. Acad. Sci. U. S. A. 99,

14422–14427.

Sjovall, J., 1959. On the concentration of bile acids in the human

intestine during absorption bile acids and steroids 74. Acta

Physiol. Scand. 46, 339–345.

Tannock, G.W., 1995. Microecology of the gastrointestinal tract in

relation to lactic acid bacteria. Int. Dairy J. 5, 1059–1070.

Tannock, G.W., 1999. A fresh look at the intestinal microflora. In:

Tannock, G.W. (Ed.), Probiotics: A Critical Review. Horizon

Scientific Press, Wymondham, England, pp. 5–14.

Tochiura, T., Sakai, K., Fujiyoshi, T., Tachiki, T., Kumagai, H.,

1986. p-nitrophenyl glycoside-hydrolyzing activities in bifido-

bacteria and characterization of h-D-galactosidase of Bifidobac-terium longum 401. Agric. Biol. Chem. 50, 2279–2286.

Zarate, G., Gonzalez, S., Perez-Chaia, A., Oliver, G., 2000. Effect

of bile on the h-galactosidase activity of dairy propionibacteria.

Lait 80, 267–276.