Download Protective effect of the bile salt hydrolase-active

Appl Microbiol Biotechnol (2000) 53: 709±714
Ó Springer-Verlag 2000
ORIGINAL PAPER
P. De Boever á R. Wouters á L. Verschaeve
P. Berckmans á G. Schoeters á W. Verstraete
Protective effect of the bile salt hydrolase-active
Lactobacillus reuteri against bile salt cytotoxicity
Received: 15 July 1999 / Received revision: 10 January 2000 / Accepted: 14 January 2000
Abstract Bacterial bile salt hydrolysis is considered a
risk factor for the development of colon cancer because
of the risk of forming harmful secondary bile salts after
an initial deconjugation step. In this study, the in¯uence
of enhanced bacterial bile salt transformation by the bile
salt hydrolase-active Lactobacillus reuteri was studied in
batch culture using the microbial suspension of the
Simulator of the Human Intestinal Microbial Ecosystem;
(SHIME), which was supplemented with oxgall at 5 g/l
or 30 g/l. Changes in the fermentative capacity of the
microbial ecosystem and the (geno)toxic properties of
the SHIME supernatants were investigated. Increasing
concentrations of oxgall inhibited the fermentation.
Transient cell toxicity was observed for samples supplemented with 5 g oxgall/l, while samples with 30 g oxgall/l
exhibited toxicity. The results of the haemolysis test
suggest that the detrimental e€ects were probably due to
the membrane-damaging e€ects of bile salts. In all cases,
the adverse e€ects could be counteracted by the addition
of 7.5 ‹ 0.5 log10 CFU L. reuteri/ml. Plausible mechanisms for the protective properties of L. reuteri could
involve a precipitation of the deconjugated bile salts and
a physical binding of bile salts by the bacterium, thereby
making the harmful bile salts less bioavailable.
Introduction
Colorectal cancer is the second most common cause of
cancer deaths in most developed countries (Ghadirian
P. De Boever á R. Wouters á W. Verstraete (&)
Laboratory of Microbial Ecology and Technology,
Faculty of Agricultural and Applied Biological Sciences,
University Ghent, Coupure links 653, 9000 Gent, Belgium
e-mail: [email protected]
Tel.: +32-9-264/59/76
Fax: +32-9-264/62/48
L. Verschaeve á P. Berckmans á G. Schoeters
Environmental Toxicology,
Flemish Institute for Technological Research (VITO),
Boeretang 200, 2400 Mol, Belgium
et al. 1997). Although there have been advances in
radiotherapy, chemotherapy and surgery, mortality
caused by colorectal cancer remains very high and has
decreased only slightly. Thirty percent of all colon cancer deaths can be linked to diet (Stone and Papas 1997).
Probably one of the most cited mechanisms is that of a
high-fat diet. To resorb the increased amount of dietary
fat, more conjugated primary bile salts are secreted in
the small intestine. This leads to an increased ¯ux of bile
salts to the colon and an increased metabolism of the
bile salts by the indigenous ¯ora (Giovannucci and
Goldin 1997). Some of the bile salts generated by the
micro-organisms have been incriminated in colonic
carcinogenesis (Singh et al. 1997).
One of the most important bacterial bile salt transformations is bile salt hydrolysis, which is mediated by a
wide range of colon anaerobes (Hill 1995). The bile salt
hydrolase enzyme releases the glycine and/or taurine
moiety from the side chain of the bile salt steroid core
and generates deconjugated primary bile salts, which are
less water-soluble and are excreted more easily via the
faeces (De Smet et al. 1994). The principle of bile salt
hydrolysis was used to decrease the serum cholesterol in
pigs through interaction with the host's bile salt
metabolism (De Smet et al. 1998). In the latter, the
bacterial bile salt hydrolase activity in pigs was enhanced
by oral administration of Lactobacillus reuteri. This
caused a greater drain on the bile salt pool, resulting in a
loss of feedback inhibition on bile salt synthesis and an
increased conversion of cholesterol into bile salts.
Because of this, the serum cholesterol levels in the pigs
were lowered. During this in vivo experiment, no
detailed information was collected about the possible
probiotic e€ect of this strain. An in vitro study using the
Simulator of the Human Intestinal Microbial Ecosystem
(SHIME) was carried out recently to evaluate the e€ect
of adding L. reuteri on the composition and activity of
the gut microbiota (Nollet et al. 1999). The experiments
indicated that L. reuteri was able to repress the concentration of Enterobacteriac and coliforms, which are
considered potential enteropathogens (Isolauri et al.
710
1991). It was suggested that the competitiveness of
L. reuteri was largely due to its bile salt hydrolase
activity (Nollet et al. 1999).
Although the bile salt hydrolase-active L. reuteri
o€ers potential as a `biological' alternative to pharmaceutical interventions to treat hypercholesterolaemia and
to be used as a probiotic, there is medical concern about
possible side e€ects. According to some authors
(Hylemon and Glass 1983; Kandell and Bernstein 1991),
enhanced bile salt hydrolytic activity is not favourable
because a subsequent dehydroxylation of these deconjugated primary bile salts by 7a-dehydroxylase-active
strains could generate toxic and/or mutagenic secondary
bile salts (Marteau et al. 1995; Nagengast et al. 1995).
It was suggested that an increase in faecal secondary bile
salts upon ingestion of bile salt hydrolase-active lactobacilli should be regarded as a potential long-term colon
cancer-promoting e€ect (van Faassen et al. 1987).
Furthermore, these toxic bile salts could disturb the
normal microbiota of the gut, leading to diarrhoea,
mucosal in¯ammation or activation of harmful drugs
and carcinogens in the intestinal contents (Salminen
et al. 1996).
This study investigated the e€ects of enhanced
bacterial bile salt modi®cations, induced by adding
L. reuteri, on the fermentative capacity of the simulated
microbiota of the colon. This was done by analysing the
concentration of short chain fatty acids (SCFAs) and the
b-galactosidase activity. The possible generation of
harmful bile salts, induced by enhanced bacterial bile
hydrolase activity was investigated using a haemolysis
test, Vitotox and the neutral red uptake (NRU) cytotoxicity assay.
Materials and methods
Simulator of the Human Intestinal Microbial Ecosystem
The possible adverse e€ects linked to bile salts and/or enhanced
bacterial bile salt hydrolase activity were investigated in batch
experiments using the gut microbiota cultured in the SHIME.
This six-stage computer-controlled reactor was developed to
simulate the bacterial communities found in the distinctive parts
of the human intestine. Each of the six reactors contains the
microbiota of a di€erent part of the human gastro-intestinal tract,
in sequence (compartment 1±6): the stomach, the duodenum, the
small intestine, the ascending, the transverse and the descending
colon (Molly et al. 1993). The reactor was set up and validated as
described previously (Molly et al. 1994). Brie¯y, the last three
vessels were inoculated on ®ve consecutive days with a faecal
suspension from ®ve healthy volunteers. This inoculum was stabilised by being fed three times a day with 200 ml of a carbohydrate-based medium containing arabinogalactan (1 g/l), pectin
(2 g/l), xylan (1 g/l), starch (3 g/l), glucose (0.4 g/l) and mucin
(4 g/l).
Isolation and identi®cation
The strain was originally isolated from pig faeces and selected
because of its high bile salt hydrolase activity. The bacterium was
identi®ed as L. reuteri using SDS-PAGE analysis (De Smet et al.
1998).
Preparation of the L. reuteri culture
A 1% inoculum of the frozen L. reuteri strain was thawed and
suspended in 10 ml de Man Rogosa and Sharpe (MRS) broth
(Oxoid), which was reduced by adding 1 g/l sodium thioglycolate
(Fluka). After 24 h incubation at 37 °C, the culture was centrifuged
(10 min at 5,000 g) and washed with sterile physiological solution
before use in the batch incubations. The number of colony-forming
units (CFU) used in the test was determined by making ten-fold
serial dilutions of the overnight culture in physiological solution
(8.5 g NaCl/l) and plating the dilutions on Rogosa agar (Oxoid)
supplemented with 5 mM taurodeoxycholic acid (Dashkevicz
and Feighner 1989). The bile salt hydrolase activity was quanti®ed
by measuring the rate with which glycocholic acid was hydrolysed
using a technique described in detail elsewhere (De Smet et al.
1994).
Experimental set-up
Samples (10 ml) were withdrawn from compartment 5 of the
SHIME reactor and centrifuged for 10 min at 5,000 g. The microbial pellet was washed with an aliquot of sterile physiological
solution and resuspended in 10 ml of fresh SHIME feed, kept at
pH 7.0 by means of a 0.1 M phosphate bu€er. The resuspended
pellets were supplemented with 0, 5 or 30 g oxgall/l by adding an
appropriate amount of a 100 g/l ®ltersterile oxgall solution (Difco).
To investigate the e€ects of enhanced bile salt hydrolase activity, a
treatment was imposed by adding L. reuteri, resulting in a viable
concentration in the test of 7.5 ‹ 0.5 log10 CFU/ml. A series
without the oxgall addition was used as a control. Finally, the tubes
were made anaerobic by ¯ushing the liquid phase for 10 min with
oxygen-free nitrogen gas.
After 24 h incubation at 37 °C, samples were taken to determine the concentration of SCFAs and b-galactosidase activity. The
remaining liquid was centrifuged for 10 min at 5,000 g and the
supernatant was sterilised using a 0.22-lm ®lter. The sterile culture
supernatants were analysed for possible (geno)toxic products by
means of the haemolysis test, Vitotox (Vito, Belgium) and the
NRU cytotoxicity assay.
Determination of SCFAs
SCFAs produced by the bacterial cultures were extracted and
measured with a gas chromatograph (GC; Carlo Erba Fractovap
4160) equipped with a ¯ame ionisation detector and a Delsi
Nermag integrator (Nollet et al. 1999). The GC was equipped with
a capillary free fatty acids packed column (25 m ´ 0.53 mm; ®lm
thickness 1.2 lM). Nitrogen was used as a carrier gas at a ¯ow rate
of 20 ml/min. The column temperature was 130 °C and the
temperature of injection port and detector was 195 °C. The concentration of the SCFAs was expressed in millimoles per liter.
Determination of b-galactosidase activity
The samples were centrifuged at 10,000 g for 10 min. Cell-free
supernatant (100 ll) was pipetted into a 96-well plate, with 100 ll
of 5.0 mM p-nitrophenyl-b-galactopyranoside, prepared in a 0.1 M
phosphate bu€er (pH 6.5). The plates were incubated at 37 °C and
the absorbance at 405 nm was read after 30 min with a Biokinetics
EL312e multi-well reader. The amount of p-nitrophenol released
was measured based on a standard curve of p-nitrophenol. The
results were expressed in micromoles of p-nitrophenol released per
ml per min (Berg et al. 1978).
Determination of haemolysis
Part of the ®ltersterile supernatant (20 ll) was mixed with 140 ll
phosphate-bu€ered saline. After incubation for 1 min at 37 °C in a
711
shaking water bath, 40 ll of red blood cells (10% suspension, ICN)
were added. Simultaneously, red blood cells were incubated in
phosphate-bu€ered saline (0% lysis) and in double-distilled water
(100% lysis). Samples were centrifuged for 1 min at 10,000 g after
incubation for 10 min at 37 °C. The supernatant was diluted four
times in double-distilled water and percentage haemolysis was
determined by measuring the absorption at 540 nm (Van Der Meer
et al. 1991).
(Geno)toxicity evaluated with Vitotox
This newly developed genotoxicity test employs Salmonella
typhimurium TA104 recN. This strain contains the lux operon
under transcriptional control of the recN gene, which is a part of
the SOS-system. Incubation of the strain with a genotoxic solution results in a derepression of the recN promotor and an
increased expression of the lux operon, resulting in increased light
production. Some products act on the light production or
enhance the bacterial metabolism, creating false positive results.
Therefore, a S. typhimurium TA104 with a promoterless lux
operon (pri) was used as a control. This constitutively light-producing strain can also be used as a toxicity sensor, with a decrease
in light production being interpreted as an indication of toxicity
(van der Lelie et al. 1996).
S. typhimurium TA104 recN and pri were grown overnight on
a rotative shaker (170 rpm) at 37 °C in a normal bacterial growth
medium supplemented with CaCl2. Next, the bacterial suspensions
were diluted 500 times in 2.5 ml fresh growth medium. An aliquot
of these dilutions (90 ll) was added to each well of a 96-well
plate, already containing 10 ll SHIME supernatant diluted ten
times. The plates were placed in a Microlumat LB96P luminometer (EG and G Berthold) and the light production of the strains
was followed as a function of time, using the following measuring
conditions: 1 s/well, cycle time of 300 s, 4 h measuring time,
30 °C.
The validity of the test was veri®ed using a known genotoxic
compound [0.02 lmol 4-nitroquinoline-1-oxide/l dimethylsulphoxide (DMSO)]. DMSO was used as a negative control. After
completion of the measurements the signal to noise ratios (S/N)
were plotted as a function of the incubation time. In the case of
genotoxic e€ects, the ratio of the maximum S/N of the recN strain
versus the maximum S/N of the pri strain (rec/pri) became larger
than 2. In the case of toxic e€ects, the maximum S/N for the pri
strain became smaller than 0.8.
Toxicity evaluated with the NRU assay
HeLa cells (ECECCC 85060701) were harvested at subcon¯uency
(70±95%) and plated in 96-well plates. Each well contained 200 ll
tissue culture medium (TCM) with 2,000 cells. After 24 h incubation at 5% CO2, 37 °C and 95% humidity, the TCM was
removed and a TCM containing either sterile SHIME culture
supernatants or a toxic compound (0.2 lM cycloheximide)
was added. Sterile water was used as a negative control.
The SHIME liquids were tested in six di€erent concentrations at
0.04±1.25%.
After 3 days incubation, the TCM was removed and replaced
with a prewarmed TCM containing 1% neutral red. After a further
3 h incubation, the NRU by the HeLa cells was measured using a
Cyto¯uor 2350 (PerSeptive Biosystems). The NRU of the cells incubated with diluted SHIME suspensions or cycloheximide was
expressed as a percentage compared to the NRU of the negative
control, with a decreased NRU indicating toxicity (Vander Plaetse
and Schoeters 1995).
Statistical analysis
Statistical analysis of the experiments were performed using an
unpaired two tailed t-test.
Results
Fermentation capacity of the colonic microbial
ecosystem
SCFAs are end-products of bacterial metabolism and a
concentration decrease in comparison with the control is
an indication of inhibition of bacterial activity over the
total incubation period. Measuring enzyme activity after
24 h provides information about the bacterial activity at
the end of the incubation period. In contrast with the
measurement of SCFAs, it only re¯ects bacterial activity
at the sampling time. The data about the concentration
of SCFAs and b-galactosidase activity indicate that an
oxgall supplementation burdened the fermentation of the
gut microbiota in a concentration-dependent manner
(Table 1). The concentration of SCFAs was lower in
comparison with the control (0 g/l oxgall + L. reuteri),
although not signi®cantly when oxgall at 5 g/l or 30 g/l
was added. Adding L. reuteri led to an increased
concentration of SCFAs, which was only statistically
di€erent from the control (P £ 0.01) in the case of 5 g
oxgall/l (Table 1). A decrease in b-galactosidase activity
was noticed with increasing concentrations of oxgall.
This reduced activity was completely (in the case of 5 g
oxgall/l) or partially (in the case of 30 g oxgall/l) counterbalanced by the addition of L. reuteri (Table 1). When
the ratio for SCFAs production and b-galactosidase activity was calculated between the series with L. reuteri
and the series without L. reuteri, it was observed that in
all cases the values were higher than 1. This indicates that
the addition of L. reuteri circumvented the negative effects of the oxgall supplementation. The presence of
7.5 ‹ 0.5 log10 CFU L. reuteri/ml in the test was determined using the plating data of the overnight L. reuteri
culture. The quantitative determination of the bile salt
hydrolase activity indicated that cholic acid was released
at a rate of 2.56 lmol per 1010 CFU per min.
Haemolytic e€ects
The haemolytic capacity of the SHIME supernatants
was determined after 24 h incubation. Samples without
Table 1 Short chain fatty acids (SCFAs) production (lmol/ml) and
b-galactosidase activity (lmol p-nitrophenol released/ml per min)
in Simulator of the Human Intestinal Microbial Ecosystem suspensions after 24 h incubation with increasing concentrations of
oxgall and with or without 7.5 ‹ 0.5 log10 CFU Lactobacillus
reuteri/ml (n = 3). Signi®cant di€erence from the series 0 g
oxgall/l + L. reuteri: *P £ 0.05, **P £ 0.01
Series
Parameter
SCFAs
0 g oxgall/l + L. reuteri
5 g oxgall/l
5 g oxgall/l + L. reuteri
30 g oxgall/l
30 g oxgall/l + L. reuteri
58.2
55.8
68.8
53.3
63.1
‹
‹
‹
‹
‹
b-galactosidase
0.7
0.4
0.5**
1.1
3.2
171.3
134.6
190.4
87.6
104.4
‹
‹
‹
‹
‹
6.2
2.8*
6.7
3.5**
5.0*
712
oxgall and L. reuteri treatment did not induce lysis of the
red blood cells. The supernatants originating from the
liquids supplemented with 5 g oxgall/l caused total lysis
(100 ‹ 12%) during the 10 min incubation. The addition of L. reuteri reduced the lytic e€ect to 2.1 ‹ 2.8%.
All samples caused total lysis when liquids with 30 g
oxgall/l were tested, even when L. reuteri was added.
(Geno)toxicity evaluated with Vitotox
In the Vitotox assay, the reference substance nitroquinoline-1-oxide brought about a rec/pri signal of
5.05 ‹ 0.61, indicating genotoxicity. Without the
L. reuteri supplement, the rec/pri ratios were 1.13 ‹
0.09 and 1.01 ‹ 0.09 for 5 g oxgall/l and 30 g oxgall/l
respectively. When L. reuteri was added, the ratios were
1.04 ‹ 0.35 and 0.92 ‹ 0.04 respectively. The data indicate that none of the sterile SHIME culture supernatants exhibited genotoxic e€ects. The S/N ratio for the
pri strain became lower than 0.8 when 5 g oxgall/l was
added, indicating physiological cell toxicity. The pri
strain was able to recover from this toxic shock as the
maximum S/N became higher than 1 during the test.
The addition of L. reuteri prevented this toxic e€ect and
temporarily stimulated the light production of the test
strain. In the case of 30 g oxgall/l, the Salmonella strain
was only able to recover when the supernatant originated from a series supplemented with 7.5 ‹ 0.5 log10
CFU L. reuteri/ml (Fig. 1).
Toxicity evaluated with the NRU assay
The NRU assay was validated by verifying that 0.2 lM
cycloheximide induced an inhibition of NRU by HeLa
Fig. 1 Kinetics of the Signal/Noise ratio of the Vitotox Salmonella
typhimurium TA104 pri strain incubated with sterile Simulator of the
Human Intestinal Microbial Ecosystem (SHIME) supernatants
supplemented with 5 g oxgall/l, with (s) or without (d) 7.5 ‹ 0.5
log10 CFU Lactobacillus reuteri/ml and 30 g oxgall/l with (,) or
without 7.5 ‹ 0.5 log10 CFU L. reuteri/ml (.)
cells of 9±18%. No di€erence in NRU was seen between
the control and the series incubated with dilutions of
®ltersterile SHIME supernatants supplemented with
5 g oxgall/l with and without the bile salt hydrolaseactive L. reuteri (results not shown). When the highest
three dilutions (0.31, 0.62 and 1.25%) of the series
supplemented with 30 g oxgall/l were tested, a reduced
NRU by the HeLa cells could be observed. This e€ect
was not observed when the suspension was preincubated
with 7.5 ‹ 0.5 log10 CFU L. reuteri/ml (Fig. 2).
Discussion
In a faecal suspension simulated by the SHIME reactor
(Molly et al. 1994), we investigated whether enhancement of bacterial bile salt modi®cation, by the addition
of the bile salt hydrolase-active L. reuteri, would lead to
increased dehydroxylation and the generation of harmful bile salts. The SHIME liquids (with and without
L. reuteri) were incubated for 24 h with 5 g oxgall/l and
30 g oxgall/l. According to the manufacturer of the
oxgall, the concentrations supplied to the suspensions
corresponded to about 1 mM and 6 mM bile salts respectively. Addition of 7.5 ‹ 0.5 log10 CFU L. reuteri/ml
to the test tubes with oxgall at 5 g/l and 30 g/l counteracted the detrimental e€ects. The SCFAs concentration increased to levels higher than the control, while the
b-galactosidase activity recovered only partially. The
results of the haemolysis test suggest that the detrimental
e€ects of adding oxgall were probably due to membranedamaging e€ects. Any lysis of the erythrocytes used in
this test can be attributed to membrane-damaging e€ects
as these cells do not possess cell organelles and lack
speci®c mechanisms for the uptake and metabolism of
bile salts (Pazzi et al. 1997). Due to the L. reuteri addition, haemolysis disappeared almost completely in the
case of 5 g oxgall/l.
Fig. 2 Neutral red uptake (%NRU) by HeLa cells treated with sterile
SHIME culture supernatants supplemented with 30 g oxgall/l (d) and
30 g oxgall/l with 7.5 ‹ 0.5 log10 CFU L. reuteri/ml (s). Data are the
mean values for two separate experiments. Error bars indicate
standard deviations. Signi®cant di€erence from the series without
L. reuteri: **P £ 0.01
713
The potential generation of genotoxic bile salts after
an enhanced bile salt hydrolysis was investigated with
Vitotox. None of the samples revealed genotoxic e€ects.
At the concentration of 5 g oxgall/l, all samples exhibited transient toxicity on the Vitotox strain. When the
solutions with 30 g oxgall/l were tested, the Vitotox
strain only recovered from the toxic pulse when the
solution was preincubated with L. reuteri. Possible
cytotoxic e€ects of the SHIME supernatants were traced
using the NRU cytotoxicity assay. The HeLa cells
employed in this test are human epithelial cells derived
from a cervix carcinoma. They are non-specialised cells,
suitable for measuring general toxicity caused by substances interfering with non-specialised housekeeping
cell functions. Again, increasing concentrations of oxgall
caused detrimental e€ects and these were circumvented
by the addition of L. reuteri.
It is currently thought that the promotion of colon
cancer by dietary fat involves the excess production of
bile salts and the bacterial conversion of conjugated
primary bile salts to potentially dangerous unconjugated
secondary bile salts (Morotomi et al. 1997). Intestinal
bacteria ®rst deconjugate bile salts before they are further metabolised (Batta et al. 1990). The predominant
metabolism of the deconjugated primary bile salts (cholic
acid and chenodeoxycholic acid) is the 7a-dehydroxylation into the secondary bile salts (deoxycholic acid and
litocholic acid). Because of the risk of forming harmful
secondary bile salts after an initial deconjugation step,
bacterial bile salt hydrolysis has been considered a risk
factor for the development of colon cancer. The precise
mechanism through which these molecules exert their
e€ect is not fully understood. On one hand, it is thought
that they might act through co-mutagenic and co-carcinogenic e€ects, thereby enhancing colorectal neoplasm
(Martin et al. 1981). On the other hand, the hydrophobic
nature of the secondary bile salts could be responsible for
disrupting the membrane integrity of mucosal cells, ultimately leading to cytotoxicity. This would increase cell
proliferation, which is considered to be a biomarker for
the development of cancer (Preston-Martin et al. 1990).
There are numerous examples that bile salt hydrophobicity correlates well with toxic e€ects on all kinds of cells:
bacterial cells, isolated hepatocytes, gastric mucosa, colonic mucosa, etc. (llani and Granoth 1990; Heuman
et al. 1996; Pazzi et al. 1997). In the case of toxicity on
bacterial cells, De Smet et al. (1995) suggested that the
protonated form of the bile salt exhibits its e€ect through
the same mechanism as organic acids, namely by causing
intracellular acidi®cation. The conjugated bile salts,
which are protonated, enter the cell via passive di€usion
and are dissociated in the bacterial cytoplasm due to the
higher pH. This causes acidi®cation of the cytoplasm and
collapse of the proton motive force, resulting in an inhibition of the nutrient transport and thus toxicity.
The results presented in this paper clearly indicate
that bile salts, supplemented under the form of oxgall
at millimolar concentrations, cause toxic e€ects on
bacterial cells, blood cells and epithelial cells. In par-
ticular, the data from the red blood cells lead to the
reasonable assumption that the mode of action is toxicity through membrane damage. This phenomenon has
been reported in literature (Albalak et al. 1996; Shekels
et al. 1996). Based on literature data, it was assumed
that enhanced bile salt hydrolysis would make more
deconjugated bile salts available for 7a-dehydroxylation.
This would lead to a higher concentration of secondary
bile salts which are (geno)toxic and mutagenic compounds (Nagengast et al. 1995). Most of this evidence
was gained from in vitro tests and laboratory animal
models. In the latter, secondary bile salts have been
shown to induce hyperproliferation of the colonic
epithelium in rats, which makes the animals more susceptible to experimentally induced colon cancer (Christl
et al. 1995). However, this hypothesis is nevertheless
considered controversial because of contradictory case
control studies (Owen 1997).
In our experiments, addition of the bile salt hydrolase-active L. reuteri did not result in an increase in
detrimental e€ects, but on the contrary brought protection against the bile salts. Based on the bile salt
hydrolase data, it was estimated that during the 24 h
incubation period about 1 mmol/l glycocholic acid was
hydrolysed. If glycocholic acid was the sole bile salt
present in oxgall, then approximately 100% and 20%
would have been hydrolysed in the tubes to which 5 g
oxgall/l and 30 g oxgall/l was added. A plausible
mechanism by which detrimental e€ects of bile salts
could have been avoided is precipitation of the deconjugated bile salts, thereby decreasing the bioavailable
concentration. It is well known that bile salt transformations have an impact on physicochemical properties
such as ionisation, solubility and micelle formation,
which are correlated with the structure of the bile salt
molecule. Removal of the amino acid moiety from the
side chain by deconjugation results in compounds that
are less resistant to precipitation at low pH or by
divalent cations such as Ca2+ (Fini and Roda 1987;
De Boever and Verstraete 1999). A second mechanism
for the protective properties of L. reuteri could involve a
physical binding of bile salts by the bacterium, thereby
making them less bioavailable. Evidence from in vitro as
well as in vivo studies supports the concept that Lactobacilli and other lactic acid bacteria are able to bind a
wide range of mutagens and (geno)toxins (Hosoda et al.
1992; Orrhage et al. 1994; Pool-Zobel et al. 1996).
All tests were done with a simulator of the gastrointestinal microbial ecosystem (SHIME). Therefore, these
in vitro studies should be considered as a preliminary
evaluation of the putative protective action exerted by
L. reuteri when it is consumed as a probiotic. More
profound research has to be conducted about the type of
bile salt removed from the liquid phase and the precise
mechanism involved in this process. Nevertheless, the
potential decrease of the bioavailable concentration of
toxic bile salts is a point in favour of the use of L. reuteri
as a human probiotic (Salminen et al. 1996; De Smet
et al. 1998; Nollet et al. 1999).
714
Acknowledgements This research has been funded by a scholarship
from the Flemish Institute for the Improvement of Scienti®c±
Technological Research in Industry (IWT). The experiments
carried out at the VITO were sponsored by AVECOM (Belgium).
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