Food and Chemical Toxicology 47 (2009) 1064–1068

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Food and Chemical Toxicology

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Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1

A. Hernandez-Mendoza a, H.S. Garcia a, J.L. Steele b,*
a
UNIDA-Instituto Tecnológico de Veracruz, M.A. de Quevedo 2779, Veracruz, Ver. 91897, Mexico
b
Department of Food Science, 1605 Linden Dr., University of Wisconsin, Madison, WI 53706, United States

a r t i c l e i n f o a b s t r a c t

Article history: It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading food-
Received 7 August 2008 borne carcinogens would reduce human exposure to these deleterious compounds. In the present study,
Accepted 27 January 2009 the ability of eight strains of Lactobacillus casei to bind aflatoxin B1 in aqueous solution was investigated.
Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B1 binding was
assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage,
Keywords: human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain
Mycotoxins
with the highest AFB1 binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 lg/mL).
Lactobacillus casei
Aflatoxin probiotic
In general, the human isolates bound the most aflatoxin B1 and the cheese isolates the least. Stability of
Bile salts the bacterial–aflatoxin complex was assessed by repeated washings. Binding was to a limited degree
(0.6–9.2% release) reversible; the L. casei 7R1–aflatoxin B1 complex exhibited the greatest stability. L. casei
L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the
bacterial cells to bile significant increased aflatoxin B1 binding and the differences between the strains
was reduced.
Ó 2009 Published by Elsevier Ltd.

1. Introduction physical, chemical, and biological methods. Physical methods in-

The human diet contains a wide variety of natural carcinogens
that are present in foods as the result of contaminated raw mate-
rials or produced during the processing and/or cooking of foods.
Aflatoxins (AFs), a group of potent mycotoxins with mutagenic,
carcinogenic, teratogenic, hepatotoxic and immunosuppressive
properties, are of particular importance because of their adverse
effects on animal and human health (Lewis et al., 2005). AFs are
produced as secondary metabolites of fungal strains (Aspergillus
flavus, Aspergillus parasiticus and Aspergillus nomius) that grow on
a variety of food and feed commodities during growth, harvest,
storage, and transportation of these products (Peltonen et al.,
2001; Jiang et al., 2005). Aflatoxin B1 (AFB1), the most toxic AF, is
of particular interest because it is a frequent contaminant of many
food products and one of the most potent naturally occurring
mutagens and carcinogens known (Teniola et al., 2005).
Concerns related to the negative health impacts of AFs have
lead to the investigation of strategies to prevent their formation
in foods, as well as, to eliminate, inactivate or reduce the bioavail-
ability of these toxins in contaminated products. Techniques to
eliminate, inactivate or reduce the bioavailability of AFs include
Abbreviations: AFB1, aflatoxin B1; AFs, aflatoxins; CFU/mL, colony-forming units
per milliliters; LAB, lactic acid bacteria; PBS, phosphate-buffer saline.
* Corresponding author. Tel.: +1 (608) 262 5960; fax: +1 (608) 262 6872.
E-mail addresses: adrianqfb@yahoo.com (A. Hernandez-Mendoza), hsgarcia@
itver.edu.mx (H.S. Garcia), jlsteele@wisc.edu, hugosgg@gmail.com (J.L. Steele).
clude manually sorting out of contaminated grains (detected by
fluorescence) and removal of contaminated grains or kernels via
flotation. These processes have at least two drawbacks: (1) high
cost of removing and disposing of the contaminated materials,
and (2) difficulty achieving complete removal contaminated mate-
rials without wasting significant portions of uncontaminated prod-
uct (Yiannikouris and Jouany, 2002). Chemical methods, which
alter the chemical structure of AFs by a chemical treatment (e.g.
ammonia gas or ammonium hydroxide, 0.10% sodium hydroxide
solution liquid, propionic acid solution bubbled with sulfur dioxide
gas), or by physical absorption onto a reactive substrate (e.g. phyl-
losilicate clay) have been used to convert AFs to less toxic and
mutagenic compounds or to immobilize them (Méndez-Albores
et al., 2007). Limitations such as loss of product nutritional and
sensory qualities, as well as, the expensive equipment required
for these techniques has encouraged the recent emphasis on bio-
logical methods (Teniola et al., 2005).
Lactic acid bacteria (LAB) and bifidobacteria, due in large part to
their GRAS status and use as probiotics, are of particular interest
for reducing the bioavailability of AFs. A number of studies have
screened these microorganisms for the ability to bind to AFs and
have reported a wide range of genus, species and strain specific
binding capacities (Bolognani et al., 1997; El-Nezami et al.,
1998b; Peltonen et al., 2000, 2001; Haskard et al., 2001; Lee
et al., 2003; Hwang et al., 2005; Zinedine et al., 2005; Shahin,
2007). The bacterial cell envelope appears to be the site of AF bind-
ing, with cell wall polysaccharides and peptidoglycan thought be

0278-6915/$ - see front matter Ó 2009 Published by Elsevier Ltd.

doi:10.1016/j.fct.2009.01.042
 A. Hernandez-Mendoza et al. / Food and Chemical Toxicology 47 (2009) 1064–1068 1065

the molecules of greatest importance (Peltonen et al., 2001; Has-
kard et al., 2001; Lahtinen et al., 2004). The stability of the AF–bac-
terial cell complex is also a key consideration when evaluating a
strains ability to reduce AF bioavailability in foods, as AF release
during gastric passage would have clear negative health implica-
tions. Binding of AF to bacterial cells has been demonstrated to
be reversible and the stability of the AF–bacterial cell complex
dependent on the strain utilized, conditions utilized during com-
plex formation and the treatment used to assess stability (Haskard
et al., 2001). The conditions of particular interest are low pH and
the presence of bile, as they are important environmental stresses
that bacterial cells encounter during passage through the gastroin-
testinal tract (GI). While the influence of low pH on AF binding has
been evaluated (Haskard et al., 2000, 2001), we are unaware of any
studies that have evaluated the influence of bile on AF binding. The
use of LAB and bifidobacteria to reduced human exposure to AFs
holds great promise, however additional studies are required to
precisely define the AF binding site(s), as well as GI relevant condi-
tions that effect AF binding and the stability of the AF–bacterial cell
complex.
A focus in our research group is the development of probiotic
strains of Lactobacillus casei. We have chosen to focus on L. casei
due to its long history of use as a probiotic, the availability of a
bank of strains with documented genetic diversity isolated from
various ecological niches, and the availability of genetic tools to
examine mechanisms of probiotic activity. The objectives of this
study were to examine a bank of strains of L. casei from different
ecological niches for the ability to stably bind AFB1 and the effect
of bile on AFB1 binding.
2. Materials and methods
2.1. Bacterial strains, growth medium and cultural conditions
L. casei strains were obtained from Dr. Steele’s culture collection at the Univer-
sity of Wisconsin, Department of Food Science (Madison, WI, USA). All of the strains
utilized were confirmed as L. casei by 16S sequencing and have been previously de-
scribed by Cai et al. (2007). The strains and their ecological niches of isolation are
listed in Table 1. Stock cultures were maintained at 80 °C in 20% (v/v) glycerol.
Working cultures were prepared from frozen stocks by two transfers in MRS broth
(De Man, Rogosa & Sharpe, DifcoTM) with inoculations at 0.3% (v/v) and static incu-
bation at 37 °C for 12 and 8 h, respectively.
To conduct AFB1-binding assays, 1% inoculations were made from the working
cultures into 30 mL of MRS broth and incubated statically for 20 h at 37 °C. After
incubation, cells were collected by centrifugation (5000 rpm, 10 min, 10 °C) and
washed twice with phosphate-buffered saline (PBS, pH 7.2) and once with sterile
double-distilled H2O. Finally, the bacterial pellets were suspended in 20 mL of ster-
ile double-distilled H2O prior to use in the AFB1-binding assays. Additionally, the
bacterial cells (2–3  109 CFU/mL) were enumerated using the pour-plate method
(Vinderola et al., 2000) and the results are expressed as colony-forming units per
milliliter (CFU/mL).
To determine the effect of exposure to bile salts on growth, survival, and AFB1
binding of L. casei strains, an aliquot (1%) of each strain was incubated for 20 h at
37 °C in MRS broth (30 mL) containing 0.05, 0.10, or 0.15% (w/v) bile salts (Sigma
Chemical Co., St. Louis, MO, USA). After incubation, cells were harvested, used in
the AFB1-binding assay, and enumerated as described above.
2.2. Preparation of AFB1 working solution
AFB1 (Sigma) was dissolved in benzene-acetonitrile (93:7, v/v) to obtain an approx-
imate concentration of 2 mg/mL (Haskard et al., 2001). To prepare a working solution
of 4.6 lg/mL, PBS was added directly and the benzene-acetonitrile was evaporated by
heating in a water bath at 80 °C for 10 min. An UV–visible spectrum (SmartSpecTM Plus
spectrophotometer, BioRad) from a sample of the solution was recorded, and the actual
concentration was calculated from the Lambert–Beer equation (A = e  c  l) using the
absorbance at 354 nm and e354 = 19,800 M1 cm1. The resulting solution was trans-
ferred to a glass bottle and stored in the dark at 4 °C until used (Zinedine et al., 2005).
2.3. AFB1 binding assay
The binding assay utilized was similar to the procedure reported by Peltonen
et al. (2001). One milliliter of each active culture (suspended in 20 mL of sterile dou-
ble-distilled H2O) was centrifuged (5000 rpm, 10 min, 10 °C), the bacterial pellet
was washed twice with 1 mL of sterile double-distilled H2O, and then suspended
in 1.5 mL of the working solution of AFB1 and incubated for 4 h at 37 °C. Subse-
quently, cells were removed by centrifugation (5000 rpm, 10 min, 10 °C) and super-
natant fluid containing residual AFB1 collected and analyzed by HPLC. For each
strain, a bacterial control (bacteria suspended in PBS) and an AFB1 control (working
solution of AFB1 without bacteria) were also incubated.
To determine the stability of the bacterial–AFB1 complex, the complex was
washed three times and the amount of released AFB1 determined. The bacterial–
AFB1 complex was washed by suspending in PBS (1.5 mL), vortexed for 15 s. and
incubating at room temperature for 5 min. Then, the bacterial cells were centri-
fuged and the supernatant collected for quantification of released AFB1. This proce-
dure was replicated two additional times.
2.4. Quantification of unbound AFB1 by HPLC
Quantification of AFB1 in supernatants was conduced by a reverse-phase method
with no extraction step, essentially as described by Peltonen et al. (2001). The HPLC
system (Hewlett Packard series 1100) consisted of a pump solvent delivery system
(Quat-pump, G1322A), a model G1322A degasser, a fluorescence detector (model
F1000, Anspec Company, Inc.), a DiscoveryÒ C18 column (25 cm  4.6 mm, 5 lm,
Supelco) and a model SP4270 integrator (Spectra-Physics) for data acquisition. A
20 lL sample was injected via an autoinjector (ALS, G1329A). Microfiltered metha-
nol-acetonitrile (60:40 v/v) was used as isocratic mobile phase with a flow rate of
1 mL/min at room temperature. Aflatoxin detection was accomplished by fluores-
cence with excitation and emission wavelengths of 362 nm and 420 nm, respec-
tively. Retention time of AFB1 was approximately 4.7 min. The percentage of AFB1
bound by the bacterial suspension was calculated using the following formula:
  
AFB1 peak area of sample
% AFB1 ¼ 1   100 ð1Þ
AFB1 peak area of toxin control

Table 1
L. casei strains examined in this study.

Strain Code Origin

L. casei ATCC 334 334 Swiss-type cheese, USA
L. casei L9 L9 Human GI tract, USA
L. casei L30 L30 Human GI tract, USA
L. casei 12A 12A Corn silage, WI, USA
L. casei 21/1 21/1 Corn silage, WI, USA
L. casei 7R1 7R1 Cheese, Denmark Fig. 1. Percentage of aflatoxin B1 bound by Lactobacillus casei strains (AFB1 4.6 lg/
L. casei DPC 3968 DPC Cheese, Ireland mL, 4 h at 37 °C) and remaining bound after three washes. Treatments with
L. casei Shirota LcS Yakult, Japan different letters in each column are statistically different by each bacteria strain
(p 6 0.05).
1066 A. Hernandez-Mendoza et al. / Food and Chemical Toxicology 47 (2009) 1064–1068

Table 2
Percentage of aflatoxin B1 released by L. casei strains during the washes.

L30 12A 21/1 L9 334 7R1 LsC DPC

a b a a c d
Wash 1 6.5 (±0.7) 9.2 (±0.8) 7.1 (±0.2) 7.0 (±0.0) 5.7 (±0.0) ND 0.6 (±0.0) 2.3 (±0.1)e
Wash 2 0.4 (±0.0) ND ND ND ND ND ND ND
Wash 3 ND ND ND ND ND ND ND ND

Each value is a mean ± (standard deviation) of duplicate assays.

Different letters in each row are significantly different by each bacterial strain (p 6 0.05).
ND means not detected.

2.6. Statistical analysis

All experiments and analyses were performed in duplicate. Data analysis was
carried out by ANOVA and Tukey’s mean comparison tests using the MINITAB sta-
tistical package v. 14.1 to identify significant differences between bacterial strains.
P values 6 0.05 were considered to be significant.

3. Results

The results for ability of different strains of L. casei to bind AFB1

are presented in Fig. 1. The amount of AFB1 bound was strain spe-
cific with the percent bound ranging from 14% to 49%. The amount
of AFB1 bound by L. casei L30 was notably more than the other
strains examined. The general trend observed was that human iso-
lates bound the most AFB1 and the cheese isolates the least. The ef-
fect of washes on release of AFB1 bound to L. casei strains is
presented in Fig. 1 and Table 2. With the exception of L30, all of
the AFB1 released was detected in the first wash. The total percent
released ranged from below the limit of detection to 9.2%. After the
washes, the amount of AFB1 bound by L. casei L30 remained nota-
bly higher than the other strains examined.
The results for ability of L. casei strains to survive or grow in the
presence of different levels of bile salts are presented in Fig. 2. It is
important to note that the strains were inoculated at 1% (v/v),
hence the initial numbers were approximately 107 CFU/mL. All of Fig. 3. Percentage of aflatoxin B1 bound by Lactobacillus casei strains treated with
different bile salts levels (none, 0.05%, 0.10%, 0.15%). Treatments with different
the strains examined were able to grow in the presence of 0.05%
letters in each column are statistically different from each other within each
treatment (p 6 0.05).

bile salts. In the presence of 0.10% bile salts, only L. casei L9 was
capable of growth, no significant death or growth was observed
with L30 and 7R1, and a reduction in viable counts was observed
with the other five strains examined. In the presence of 0.15% bile
salts, no significant loss in viability was observed with L. casei L30,
while the viable counts for the other seven strains examined
declined.
The results for the influence of incubation of the bacterial cells
in the presence of bile salts on AFB1 binding are presented in Fig. 3.
With the exception of L30, growth in the presence of 0.05% bile
salts dramatically increased AFB1 binding by the L. casei strains
examined; the highest levels of AFB1 binding observed were with
L. casei 21/1 and DPC 3968. For most strains, incubation in the pres-
ence of 0.10 and 0.15% bile salts did not result in dramatic changes
in AFB1 binding, relative to that observed with the samples incu-
bated in the presence of 0.05% bile salts; however, significant
reductions in AFB1 binding was observed with L. casei 21/1 and
DPC 3968 cells incubated in the presence of 0.10% bile salts relative
to the results obtains in the presence of 0.05% bile salts and a dra-
matic increase was observed with L. casei ATCC 334 cells incubated
in the presence of 0.10% bile salts relative to cells incubated in the
presence of either 0.05% or 0.15% bile salts.

Fig. 2. Viability of Lactobacillus casei strains after incubation in MRS broth for 20 h
at 37 °C in presence of different levels of bile salts (none, 0.05%, 0.10%, 0.15%).
Treatments with different letters in each column are statistically different from
each other within each treatment (p 6 0.05).
4. Discussion
Several strategies including physical, chemical and biological
methods have been investigated to eliminate, inactivate or reduce
 A. Hernandez-Mendoza et al. / Food and Chemical Toxicology 47 (2009) 1064–1068
the bioavailability of AFs. Probiotics, living microorganisms which,
when ingested at sufficient numbers, exert a beneficial effect on
the host organism beyond inherent general nutrition (Joint FAO/
WHO, 2002; CAST, 2007), hold great promise for reducing the bio-
availability of consumed AFs. L. casei is one of the most commonly
consumed and best characterized probiotic species. The objectives
of this study were to screen eight strains of L. casei isolated from
human, plant or dairy environments for the ability to bind AFB1,
investigate the stability of the bacterial–AFB1 complexes, and to
examine the effect of bile exposure on AFB1 binding.
Eight strains of L. casei were screened for the ability to bind
AFB1, the % bound ranged from 14% to 49% (Fig. 1). Previous inves-
tigations into levels of AFB1 binding by lactobacilli and L. casei have
reported values ranging from 5% to 84% and 0.6% to 46%, respec-
tively (Bolognani et al., 1997; El-Nezami et al., 1998b; Peltonen
et al., 2000, 2001; Haskard et al., 2001; Lahtinen et al., 2004;
Hwang et al., 2005; Zinedine et al., 2005). Also it has been previ-
ously reported that bacterial concentration influences de AFB1 re-
moval. Approximately a minimum of 2–5  109 CFU/mL is
required for significant AFB1 removal (13–50%), while a concentra-
tion of 2  1010 CFU/mL is capable of reducing the AFB1 level to less
than 0.1 and 13% (Bolognani et al., 1997; El-Nezami et al., 1998b).
The effect of various AFB1 concentrations on AFB1 removal has
been also tested. The amount of AFB1 removed increased with
increasing concentration of AFB1 but the percentage removed
was not significantly different (El-Nezami et al., 1998b). According
to this, in this study there the effects of population density or dif-
ferent toxin levels on binding capacity were not determined.
Hence, the experimental conditions were selected from previous
reports (El-Nezami et al., 1998b; Peltonen et al., 2001) in order
to minimize procedural differences among studies, and according
to preliminary assays validated in our laboratory. While it is diffi-
cult to compare results of AFB1-binding levels from different stud-
ies, due to the possible impact of procedural differences. The
results observed in the current study are similar to those reported
previously. The examination of eight strains from a species is the
most extensive screening of a single species to date; the results
support the conclusion of previous studies that the ability to bind
AFB1 is a strain dependent trait. The basis for the observed strain to
strain variation is unknown, however it is likely due to differences
in either the types, numbers, or availability of AFB1-binding sites.
Although the mechanism of binding of AFs by bacterial cells is
not well understood, it is thought that the primary cellular compo-
nents involved are peptidoglycan, as well as, cell wall polysaccha-
rides and proteins (Lahtinen et al., 2004). Additionally, it has been
suggested that AFB1 is bound to the bacteria by weak, non-covalent
interactions, such as association with hydrophobic pockets on the
bacterial surface (Haskard et al., 2001; Peltonen et al., 2001). How-
ever, it is likely that multiple components are involved in aflatoxin
B1 binding (Turbic et al., 2002) and that this interaction can be af-
fected by environmental conditions.
The strain specific stability of the bacterial cell–AFB1 complex
was investigated by quantifying the amount of AFB1 released dur-
ing washes in PBS; the% of bound AFB1 released ranged from non-
detectable to 9.2%. These results suggest that at least a portion of
the bound AF is irreversibly bound. The strain to strain variation
suggests that either completely different binding sites are present
in different strains or, more likely, that there are minor differences
in similar binding sites between that strains that varies in a strain
dependent manner. The only strain which did not release any
detectable AFB1 after 3 washes was L. casei 7R1, suggesting that
the complex formed with this strain was the most stable. The abil-
ity to stably bind AFB1 in an aqueous environment is most relevant
to the use of probiotics to bind AFs in fermented foods, where the
intent is to bind AFs prior to consumption and then to maintain the
cell–AF complex during passage through the GI tract. Of the strains
1067
examined, 7R1 and LcS have the greatest potential in this
application.
The binding of AFs by the microbiota of the GI tract after ingestion
will depend on the number of microorganisms present in the various
compartments of the GI tract and their physiological state. Due to the
low pH, the number of microorganisms in the stomach is very low,
typically less than 103 bacteria CFUs per gram of contents, while
the numbers in the small intestine increase from approximately
104 in the duodenum to 107 CFUs per gram of contents at the end
of the ileum (Booijink et al., 2007). The consumption of fermented
milk containing L. casei has been shown to result in numbers of this
probiotic strain 10- to 100-fold higher in the ileum than that of the
resident microbiota (Oozeer et al., 2006). Bile acids are present at
concentrations from 0.2% to 2.0% in the human small intestine
(Hofman, 1998) and are known to have a strong antimicrobial activ-
ity against a wide variety of microorganisms, including lactobacilli
(Begley et al., 2005). Bile is known to have a significant impact on
the bacterial cell envelope, particularly on phospholipids and pro-
teins present in the membrane and the architecture of the cell surface
(Begley et al., 2005; Gunn, 2000; Leverrier et al., 2003). Additionally,
exposure to bile has been shown to alter the expression of lactobacilli
genes that encode proteins that are found in the bacterial cell enve-
lope (Bron et al., 2006; Pfeiler et al., 2007; Whitehead et al., 2008).
Therefore, to screen strains of L. casei for the ability to bind AFs during
passage through the GI tract, it is important to evaluate the influence
of bile on AF binding. The results presented in this study demonstrate
that exposure of L. casei strains to bile had a significant impact on
AFB1 binding. In general, the strains bound more AFB1 when exposed
to bile and the differences between strains were reduced. These re-
sults are particularly remarkable given that there was more than a
100-fold difference in survival between strains upon exposure to
0.15% bile. The data suggest that exposure to bile resulted in an alter-
ation in the L. casei cell envelope that enhanced AFB1 binding and
supports previous reports which suggest that viability is not essential
for AF binding (El-Nezami et al., 1998a; Haskard et al., 2001; Lee et al.,
2003; Shahin, 2007). This alteration in the cell envelope could be the
result an adaptive response to bile salt, which includes architecture
and compositional changes on the cell surface (Gunn, 2000; Leverrier
et al., 2003). It was previously cited that protein components of the
cell wall could be related to the aflatoxin binding mechanism. In this
context, it has been reported (Kim et al., 2001) that the adaptive re-
sponse to bile salt stress involves the expression of new or existing
proteins at higher levels. Additionally, the detergent character of bile
salts may also alter the conformation of surface proteins resulting in
their misfolding or denaturation (Begley et al., 2005). Therefore, this
adaptive response could have induced most likely new aflatoxin
binding sites, or increased the already existing sites, thus raising
the bacteria ability to bind aflatoxin. In addition, the presence of bile
salts in the culture medium has been reported to affect both the gly-
colipids and phospholipids fractions of the cell membrane (Kheadr,
2006). Regardless of the mechanism, exposure to bile has been dem-
onstrated to affect both the hydrophobicity and zeta potential of bac-
terial cell surfaces (Begley et al., 2005). This fact could have increased
the amount of bound aflatoxin by weak non-covalent interactions on
the bacterial surface (hydrophobic pockets).
Several probiotic bacteria have been shown to bind AFB1 effi-
ciently in vitro (El-Nezami et al., 1998b; Haskard et al., 2001;
Peltonen et al., 2001; Lee et al., 2003; Zinedine et al., 2005; Shahin,
2007), but ex vivo results are controversial (El-Nezami et al., 2000;
Gratz et al., 2005). It has been suggested that these differences may
be caused by intestinal conditions such as pH (Haskard et al., 2000,
2001) and intestinal mucus (Gratz et al., 2004). However, further
works are still needed to assess other factors which could interfere
with AFB1 binding by probiotic bacteria. In this respect, no study
has been reported dealing with the influence of bile on AF binding.
The results in this work clearly show that AFB1 binding was en-
1068 A. Hernandez-Mendoza et al. / Food and Chemical Toxicology 47 (2009) 1064–1068
hanced by bile salts. This finding could have an important biolog-
ical significance, since probiotic bacteria are normally exposed to
bile secreted in the small intestine, which suggest that probiotics
may be more capable of binding AFB1 in the intestine, and thus re-
duce AFB1 bioavailability in the gut.
5. Conclusions
The results presented support the conclusions of previous
researchers that the ability to bind AFB1 and the stability of the
bacterial cell–AFB1 complex are bacterial strain dependent traits.
Exposure of the bacterial cells to bile significant increased AFB1
binding and the differences between the strains was reduced. This
study reinforces the importance of conducting mutagen and car-
cinogen binding assays under physiologically relevant conditions.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors want to thank Prof. Jae-Hyuk Yu (Department of
Bacteriology and Genetics) and Prof. Kirk L. Parkin (Department
of Food Science) of the University of Wisconsin-Madison for pro-
viding laboratory space and equipment. Adrian Hernandez-Men-
doza was supported by a DGEST scholarship from the
government of México.
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