Vol.

63, No 2/2016
281–286
http://dx.doi.org/10.18388/abp.2015_1094
Regular paper

Probiotic preparation reduces faecal water genotoxicity and

cytotoxicity in chickens fed ochratoxin A contaminated feed
(in vivo study)*
Katarzyna Śliżewska1*, Adriana Nowak1 and Stefania Smulikowska2
1Institute of Fermentation Technology and Microbiology, Lodz University of Technology, Łódź, Poland; 2Polish Academy of Sciences,
The Kielanowski Institute of Animal Psysiology and Nutrition, Jabłonna, Poland

The aim of the present study was to assess the genotox- biological and economic consequences of the more usu-
icity and cytotoxicity of the faecal water of chickens fed al chronic intoxications caused by lower levels of toxin
ochratoxin A (OTA) contaminated feed with and with- (Rocha et al., 2014).
out probiotic preparation. The study was performed on Data published by FAO in 2001 show that 25% of
20 healthy female Ross broiler chickens divided into 4 agricultural products are contaminated with mycotoxins,
groups: control chickens — fed with non-supplemented with their type and concentration largely dependent on
feed; PP chickens — fed feed supplemented with the the climatic zone. In spite of the fact that in Europe we
probiotic preparation; OTA chickens — fed feed con- can observe less favorable conditions for the synthesis
taminated with 1 mg per kg of OTA; OTA + PP chickens of mycotoxins than e.g. in North America or Asia, the
— fed feed contaminated with 1 mg per kg of OTA and problem of mycotoxins in grains is a very important is-
supplemented with the probiotic preparation. Faecal sue also for many European countries (primarily the
water samples were collected on the 35th day of life of Scandinavian countries, the southern parts of Germany
chickens from each group. Genotoxicity was measured as well as Austria and Italy) (FAO/WHO, 2001). There-
using the comet assay, and cytotoxicity by means of fore, the contamination of animal feeds with mycotox-
MTT tests. Mean DNA damage, measured as the percent- ins is considered to be a world-wide problem (Siegel and
age of DNA in the tails of the comets, was 8.50 ± 1.10 Babusco, 2011).
for chickens fed OTA at 1 mg/kg and 6.41 ± 0.67 in the Ochratoxin A (OTA), a secondary metabolite with
controls. The supplementation of feed with the probi- teratogenic, hepatotoxic, carcinogenic and nephrotoxic
otic preparation decreased the extent of DNA damage activity in many animal species, including human beings
to 4.74 ± 0.78. In the control group of chickens the aver- (Höhler, 1998), is mainly produced by Aspergillus ochraceus
age cytotoxicity was 38.5 ± 0.5 (in MTT), while in the pro- and Penicillium viridicatum. Being ubiquitous, these moulds
biotic preparation group (PP group) it was 31.8 ± 0.7 (in can easily contaminate foodstuffs. OTA is predominant-
MTT). After supplementation of the feed with the probi- ly found in cereal grains, cereal products, legumes, oil-
otic preparation, the genotoxicity and cytotoxicity were seed, coffee beans and animal feed. Moreover, OTA has
decreased in a statistically significant manner. been identified in the tissue and organs of animals (pigs,
chicken) fed contaminated feed. The presence of OTA
Key words: ochratoxin A, probiotics, chicken, genotoxicity, cytotoxic- has also been reported in human milk. The IARC (In-
ity ternational Agency for Research in Cancer) has classified
Received: 15 July, 2015; revised: 08 September, 2015; accepted: OTA as Group 2B, a possible human carcinogen (IARC,
09 October, 2015; available on-line: 07 January, 2016 1993). In several areas of Eastern Europe, where chronic
exposure to OTA occurs, involvement of this mycotoxin
in the aetiology of cancer of the urinary system and in
INTRODUCTION kidney pathologies typical of Balkan Endemic Nephropa-
thy (BEN) has been suspected. Studies on the correla-
Mycotoxins are secondary fungal metabolites and re- tion between OTA and BEN (Puntaric et al., 2001) have
main the subject of major concern throughout the world. shown higher OTA contamination levels in cereals from
Where present, they usually occur as trace contaminants endemic areas with respect to cereals from non-endemic
in agricultural products, in concentrations ranging from areas (Muscarella et al., 2004).
nanogram to microgram quantities per gram of material. Protection against mycotoxin contamination of raw
Intensive research on mycotoxins has been conducted plant material which is to be biotechnologically pro-
for the past 35 years. The first group of mycotoxins cessed and used as animal feed focuses on its growth
which was isolated and described in 1961, in the wake requirements, harvest and storage (Fraga et al., 2007).
of several acute animal disease outbreaks in 1960, con-
sisted of aflatoxins (Goldblatt, 1969). After the discov- *
e-mail: [email protected]
ery of aflatoxins, ochratoxins were the next major group *The results were presented at the 6th International Weigl Confer-
ence on Microbiology, Gdańsk, Poland (8–10 July, 2015).
of mycotoxins identified (van der Merwe et al., 1965). It Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; DMEM, Dul-
was typical for mycotoxicoses in general that a seasonal becco’s Modified Eagle’s Minimal Essential Medium; EDTA, ethylen-
peak in the occurrence of a toxin drew attention to the ediaminetetraacetic acid; FAO, Food and Agriculture Organization
agents which triggered acute clinical diseases. Such acute of the United Nations; MTT test, reduction assay the tetrazolium
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; OTA,
intoxications can be dramatic and economically devas- ochratoxin A; PP, probiotic preparation
tating, but they constitute only a small fraction of the
282
K. Śliżewska and others 2016

However, sometimes it is very difficult to minimise the Lymphocyte isolation and cell treatment. Blood
production of toxins by moulds, especially if a crop is was obtained from young, healthy, non-smoking donors.
exposed to changeable weather conditions favourable Peripheral blood lymphocytes were isolated by centrifu-
to contamination of cereals with moulds and their toxic gation at a density gradient of Histopaque-1077 (15 min,
products. If plant material is contaminated with myco- 182 × g) (Sigma). The pellet containing peripheral blood
toxins, it should be subject to detoxification (Akande et lymphocytes was suspended in RPMI 1640 medium
al., 2006). In the case of feed, FAO accepts the methods (Sigma). The final concentration of the lymphocytes in
of eliminating toxins by means of chemical compounds each sample was adjusted to 1 × 105 cells/ml. Lympho-
and physical processes which fulfil several requirements, cytes were incubated with 10% faecal water for 1 h at
concerning e.g. the preservation of the nutritive and sen- 37oC. In addition to the dietary treatments, two addition-
sory value and physical properties of products, as well as al treatments were included in order to validate the as-
the economic justification for the decontamination pro- say. These treatments included control cells that only re-
cess (Allameh et al., 2005). ceived the RPMI 1640 medium and a treatment in which
Biological detoxication of mycotoxins in food, raw cells were treated with hydrogen peroxide at 20 µM ap-
material and concentrated feed as well as in human and plied for 10 min at 4°C.
animal organisms is a new and very promising method. Caco-2 cell culture and treatment. The cells were
Microorganisms used for the elimination of mycotoxins cultured in Roux flasks as monolayer in Dulbecco’s
include lactic acid bacteria Lactobacillus sp. and yeast Sac- Modified Eagle’s Minimal Essential Medium (DMEM,
charomyces sp. (El-Nezami et al., 2000; Shetty & Jespersen, Sigma) with the addition of 10% FBS (Gibco), 200 mM
2006). Particular attention is paid to lactic acid bacteria L-glutamine (Sigma), 25 mM HEPES (Sigma), 100 IU/
(LAB), because they can contribute to the inhibition ml penicillin (Sigma) and 100 µg/ml streptomycin (Sig-
of mould development and production of mycotoxins ma). The cells were incubated in a CO2 incubator at
(Haskard et al., 2001; Slizewska et al., 2010). 37°C in 5% CO2 for 7–10 days to become fully differ-
The aim of the present study was to assess the in- entiated. After reaching confluence, the cells were sub-
fluence of the probiotic preparation on the genotoxicity cultivated every week. The medium was changed every
and cytotoxicity of the faecal water of chickens fed OTA 2–3 days. Caco-2 cells were tripsinised with 1% trypsin-
contaminated feed with and without probiotic prepara- EDTA (Sigma) for 2 min and gently shaken off the
tion. plastic flask. The reaction was terminated by addition of
10 ml of DMEM with 10% FBS. For the removal of
trypsin, the cell suspension was transferred to a 15 ml
MATERIALS AND METHODS
Falcon tube, centrifuged (182 × g, 5 min), decanted and
resuspended in DMEM. After the determination of cell
Probiotic preparation. The probiotic preparation count and viability by trypan blue, the cells were ready
consisted of (per 1 kg): 1010 Lactobacillus cells (Lb. pa- to use.
racasei LOCK 0920, Lb. brevis LOCK 0944 and Lb. Comet assay. After incubation, the cells were cen-
brevis LOCK 0945), 106 yeast Saccharomyces cerevisiae trifuged (182 × g, 15 min, 4°C) and the comet assay was
LOCK 0140 and 50 g of Yucca Schidigera extract. The performed in alkaline conditions according to the pro-
strains were from the Collection of Industrial Microor- cedure of Singh et al. (1988) with some modifications
ganisms (LOCK), Institute of Fermentation Technology (Blasiak and Kowalik, 2000). The cells were suspended
and Microbiology, Technical University of Lodz, Poland. in 0.75% LMP agarose and layered onto slides precoated
The strains used in the study for the preparation possess with 0.5% agarose and lysed for 1 h at 4°C in a buffer
full probiotic documentation (Śliżewska et al., 2012) and consisting of 2.5 M NaCl, 1% Triton X-100, 100 mM
are licensed (Michalowski et al., 2012). EDTA and 10 mM Tris. After lysis, the slides were
Treatment of animals. The study was performed on placed in an electrophoresis unit and DNA was allowed
20 healthy female Ross broiler chickens divided into 4 to unwind for 20 minutes in an electrophoretic solution
groups, 5 animals each, kept separately at the Institute containing 300 mM NaOH and 1mM EDTA. Electro-
of Animal Physiology and Nutrition, Polish Academy of
phoresis was conducted at 4°C for 20 minutes at an
Sciences, Jabłonna, Poland. The groups were: control electric field strength of 0.73 V/cm (30 mA). Then, the
chickens – fed non-supplemented feed; PP chickens –
fed feed supplemented with the probiotic preparation (2 slides were neutralized with 0.4 mol/l Tris and stained
with 1 µg/ml DAPI (4’,6-diamidino-2-phenylindole) and
g/kg of feed between 1st and 14th day of life and 1 g/kg covered with cover slips. Comets were observed at 200 ×
until the end of the experiment); OTA chickens – fed magnification with a fluorescence microscope (Nikon,
feed contaminated with 1 mg per kg of OTA; OTA + Japan) attached to a video camera and connected to a
PP chickens – fed feed contaminated with 1 mg per kg personal computer-based image analysis system Lucia-
of OTA and supplemented with the probiotic prepara- Comet v. 4.51 (Laboratory Imaging, Prague, The Czech
tion. All experimental procedures involving animals were Republic). Fifty images were selected from each sample,
conducted according to the Polish legal regulations con- and DNA damage was measured as the percentage of
cerning experiments on animals (following a decision is- DNA in the tail of the comets. Two parallel tests with
sued by the Local Ethical Committee for Experiments aliquots of the same sample were performed for a to-
on Animals at the University of Life Sciences in War- tal of 100 cells and mean DNA damage was calculated.
saw). Each experiment was repeated three times. Comet re-
Faecal water preparation. Faecal water samples were
sults were analysed using two-way analysis of variance
collected on the 35th day of life, for each diet, in plas- (ANOVA) while a particular mode of interaction × time
tic bags, sealed and stored at –20oC until analysis. Faecal was used to compare effects evoked by chemicals at this
water samples were extracted from faeces by homogenis- mode of interaction and suitable control. As no statisti-
ing the faeces with sterile water (1:5, v/v) for 2 min fol- cally significant interaction was found, one-way analysis
lowed by centrifugation (10 700 × g, 40 min, 4oC). The su- of variance was applied. Differences between the means
pernatant fractions were distributed to 1.5 ml Eppendorf were compared using Scheffe’s multiple comparison test.
tubes and stored at –20oC prior to analysis. Results were presented as mean ± S.E.M.
Vol. 63
Probiotic preparation reduces faecal water genotoxicity and cytotoxicity in chickens 283

Analysis was performed in three inde-

Table 1. DNA damage measured as a percentage of DNA in the comet tail in pendent experiments, each conducted in
an alkaline comet assay in human peripheral blood lymphocytes induced by
faecal water of broiler chickens. triplicate.
Control PP OTA OTA + PP % cell viability = (sample O.D./control O.D.)
Chicken num- × 100%
ber
DNA [%] in comet tail ± S.E.M. % cytotoxicity = 100 – % cell viability
1 8.19 ± 1.48 6.17 ± 0.01 10.43 ± 0.64 6.79 ± 0.46 Indicate main effect and interactive terms
for the two-way ANOVA.
2 5.90 ± 1.24 4.93 ± 1.40 9.28 ± 0.86 6.27 ± 0.70 Results were presented as mean ± S.D.
3 6.90 ± 1.07 6.82 ± 1.62 8.13 ± 3.96 3.94 ± 0.46
RESULTS
4 7.49 ± 2.27 4.14 ± 0.51 3.70 ± 0.52 2.60 ± 0.48

5 4.15 ± 0.81 4.99 ± 1.46 7.78 ± 2.35 4.09 ± 0.42 Genotoxicity

Non-exposed samples (cells in RPMI
Meana ±  S.E.M. 6.41 ± 0.67 5.30* ± 0.59 8.50 ± 1.10 4.74** ± 0.78 1640, the negative control) displayed DNA
The number of cells analysed in each treatment was 100. Data are mean values
damage levels of 1.45 ± 0.84. Treatment of
(± S.E.M.) from all chickens in each group for which faecal samples were available. a the cells with 20 µM hydrogen peroxide
the results displayed are the mean of three independent experiments; *,**statistically (positive control samples) resulted in dam-
different from: *control, **OTA (1 mg/kg), ANOVA (P < 0.05). age levels of 26.36 ± 1.62 (results from three
independent experiments).
Cytotoxicity testing. Caco-2 cells were added at Supplementation of the feed with the
5 × 103 cells/well in 96 well plates and 80 µl of culture probiotic preparation only (PP — probiotic group of
medium was added into each well. The cells were then chickens) decreased the genotoxicity of faecal water
incubated at 37°C overnight in 5% CO2 to allow them compared to the control group of chickens (with no
to attach to the plates. Next, a 20 µl aliquot of the ap- supplementation) (Table 1). In the control group, DNA
propriate sample of chicken faecal water was added to damage was in the range from 4.15 ± 0.81 to 8.19 ± 1.48
each well. The cells were incubated in a CO2 incuba- (with the mean value of 6.41 ± 0.67), while in the pro-
tor at 37°C in 5% CO2 for 72 h. After incubation, the biotic preparation group of chickens (PP group) it was
cells were washed twice with PBS/EDTA, and 100 µl from 4.14 ± 0.51 to 6.82 ± 1.62 (with the mean value of
of MTT (0.5 mg/ml in PBS) was added into each well. 5.30 ± 0.59) (Table 1, Fig. 1A and B). The results were
The cells were further incubated at 37°C in 5% CO2 for statistically significant as marked in the Table 1.
3 h. After incubation, the neutral red was carefully tak- Incubation of human lymphocytes with the fae-
en off and 50 µl of desorbing solution (1% acetic acid, cal water of the chickens induced considerable DNA
50% ethanol and 49% distilled water) was added to each damage, yielding comet tails ranging from 3.70 ± 0.52
well. In the MTT test, the formazan precipitates were to 10.43 ± 0.64 (with the mean value of 8.50 ± 1.10) for
solubilized by the addition 50 µl of DMSO (Sigma). The chickens fed feed contaminated with 1 mg/kg of OTA
absorbance was measured at 550 nm using a microplate (OTA group) (Table 1 and Fig. 1C).
reader (ASYS, Biogenet). The absorbance of the control After supplementation of the feed with the probiotic
sample (Caco-2 cells in DMEM) was set at as 100% cell preparation, we observed a statistically significant de-
viability. crease in the level of DNA damage, which was in the
range from 2.60 ± 0.48 to 6.79 ± 0.67 (with the
mean value of 4.74 ± 0.78) for chickens fed with
feed contaminated with 1 mg/kg of OTA (OTA
+ PP group). Supplementation with the probiotic
preparation reduced the genotoxicity of faecal
water in chickens fed feed contaminated with 1
mg/kg of OTA (Table 1 and Fig. 1D).

Cytotoxicity
Supplementation of the feed with the probi-
otic preparation only (PP — probiotic group of
chickens) statistically decreased the cytotoxicity
of faecal water compared to the control group
of chickens (with no supplementation) (Fig. 2).
In the control group of chickens, the average
cytotoxicity was 38.5 ± 0.5, while in the probiotic
preparation group (PP group) it was 31.8 ± 0.7
(Fig. 2).
Incubation of Caco-2 cells with the faecal
water of chickens fed feed contaminated with 1
Figure 1. Representative comets of DAPI stained human blood lympho- mg/kg OTA (OTA group) induced considerable
cytes incubated for 1 h at 37oC with faecal water of broiler chickens:
(A) control groups of chickens; (B) chickens fed with fodder supplement- cytotoxicity with a mean value of 53.9 ± 0.4 for
ed with probiotic preparation; (C) chickens fed with fodder contaminated chickens fed with feed contaminated with 1 mg/
with 1 mg/kg of OTA; (D) chickens fed with fodder contaminated with 1 kg of OTA (OTA group) (Fig. 2).
mg/kg of OTA and supplemented with probiotic preparation.
284
K. Śliżewska and others 2016

In order to investigate the mechanisms which account

for the removal of mycotoxins by LAB, the effects of
viable and heat inactivated bacteria were compared in a
number of studies (Haskard et al., 2001). Additionally,
the bacteria were treated with enzymes (such as pro-
nase E and lipase) or periodate which cause alterations
in the structure of the cell walls (Fuchs et al., 2008). On
the basis of the results obtained in those experiments,
it was postulated that the removal of aflatoxin B1 and
zearalenone is due to the non-covalent binding of the
toxins to the carbohydrate moieties of the cell walls. The
detoxification of heterocyclic aromatic amines was also
explained by this mechanism (Knasmüller et al., 2001).
However, since a decrease in their toxic effects was also
seen with cytosolic preparations of LAB, it was hypothe-
sized that other mechanisms (e.g. interactions with short
chain fatty acids) may also play a role (Stidl et al., 2007).
The mechanisms involved in the cellular toxicity of
OTA are still unresolved. Distinct mechanisms such as
active transport by organic anion transporters and cel-
Figure 2. Cytotoxicity of the faecal water of chickens after feed- lular accumulation (Doorten et al., 2006), as well as the
ing with probiotic preparation and/or ochratoxin A.
Asterisks indicate significant differences (P < 0.05). The results dis- chelation of OTA with cellular Fe2+ molecules resulting
played are means (± S.D.) of three independent experiments. in an increased production of ROS, have been proposed
to mediate OTA toxicity (Schaaf et al., 2002). Cellular
After supplementation of the feed with the probiotic DNA damage has been linked in several studies to bio-
preparation, a statistically significant decrease in the cy- transformation processes, both in vitro and in vivo, as an
totoxicity of the faecal water was observed, which was increased number of DNA adducts were found in hu-
39.5 ± 0.5 for chickens fed feed contaminated with 1 mg/ man bronchial epithelial cells expressing CYP2C9 (El
kg of OTA (OTA + PP group). Adlouni et al., 2000) and kidney microsomes (Obrecht-
Pflumio and Dirheimer, 2000) as well as in mice (Obre-
DISCUSSION cht-Pflumio et al., 1996), rats and pigs (Faucet et al.,
2004) treated with OTA. OTA induced mutations on the
Biological detoxification of mycotoxins in food, raw lacZ’ gene in NIH/3T3 cells, transfected with distinct
products, mixed protein feeds and also in human and human CYP450 enzymes (de Groene et al., 1996). These
animal organisms is a relatively new and promising cells, transfected with a specific human CYP450 enzyme
method. Several microorganisms have been used to elim- and at the same time with human oxidoreductase were
inate mycotoxins: Acinetobacter calcoaceticus, Aspergillus ni- used in the experiments described here, directed to the
ger, Bifidobacterium sp., Lactobacillus sp., Enterococcus faecium, analysis of single-stranded DNA breaks (SSBs) following
Pseudomonas sp. and Saccharomyces sp. strains (Sangare et single cell electrophoresis (comet assay) (Doorten et al.,
al., 2014; Ŝtyriak et al., 2001; Topcu et al., 2010; Vindero- 2006).
la and Ritieni, 2015). Special attention should be paid to Several lines of evidence indicate that the carcinogen-
lactic acid bacteria because of their favourable influence ic action of OTA is correlated with its genotoxicity, as
on human organisms (probiotic bacteria) and widespread reflected by DNA adduct formation (Castegnaro et al.,
use in the production of fermented foods. These bacte- 1998; Pinelli et al., 1999), leading to mutagenic effects
ria inhibit the growth of moulds as well as mycotoxins (Malaveille et al., 1991; de Groene et al., 1996). DNA-
production (Madrigal-Santillán et al., 2006). xenobiotic binding is considered to be a critical step in
The aim of our study was to check if the probiotic the initiation of mutagenesis and carcinogenesis (Miller
preparation (composed of lactic acid bacteria, yeasts and & Miller, 1981). The process of chemical carcinogenesis
Yucca schidigera extract) influenced the genotoxicity and is initiated by the covalent binding of carcinogens or
cytotoxicity of the faecal water of chickens fed OTA their reactive metabolites to DNA, thus forming DNA
contaminated feed. We observed that genotoxicity of the adducts (Pinelli et al., 1999). There is a good correlation
faecal water occurred both in the presence or absence between DNA adduct formation and the frequency of
of OTA. However, after supplementation of the feed mutations (for a review see Lutz & Gaylor, 1996). To in-
with the probiotic preparation, genotoxicity decreased in teract with cellular macromolecules and thus initiate can-
a statistically significant manner. The decrease was more cer, most chemical carcinogens require metabolic activa-
pronounced in the group of chickens fed feed contami- tion (Miller & Miller, 1981), but the metabolic pathways
nated with OTA (OTA+PP). Therefore, supplementation involved in OTA toxicity and genotoxicity remain poorly
with the probiotic preparation contributes to the reduc- understood (Pinelli et al., 1999).
tion of genotoxicity of the faecal water of chickens fed There are several mechanisms for OTA toxicity at
OTA contaminated feed. The mechanism underlying this the cellular level. OTA is a competitor of phenylalanine-
effect may be associated with the ability of the probiotic tRNA ligase, thereby inhibiting protein synthesis. The
preparation to bind OTA, resulting in its decreased con- presence of phenylalanine or aspartame (an analogue of
centration in the chicken colon, although the supplemen- phenylalanine) decreases toxicity by competition with
tation of feed with the probiotic preparation only (PP) OTA (Creppy et al., 2004; Bayman & Baber, 2006). Oth-
also decreased DNA damage in the control group. After er enzymes are also affected by exposure to OTA. Other
the supplementation of feed with the probiotic prepara- mechanisms include formation of DNA adducts, apopto-
tion, a statistically significant decrease in the cytotoxicity sis, interference with the cytoskeleton, lipid peroxidation
of faecal water was observed in chickens fed feed con- and inhibition of mitochondrial respiration (Bayman &
taminated with 1 mg/kg of OTA (OTA + PP group). Baker, 2006).
Vol. 63
Probiotic preparation reduces faecal water genotoxicity and cytotoxicity in chickens 285

The cytotoxicity of ochratoxin A has been investi- Fraga M, Curvella F, Gatti M, Cavaglieri L, Dalcero A (2007) Poten-
gated by several authors and EC50 values were gener- tial aflatoxin and ochratoxin A production by Aspergillus species in
poultry feed processing. Vet Res Com 31: 343–353. DOI:10.1007/
ally observed to lie within the micromolar range. Differ- s11259-006-3434-x.
ences between the specific EC50s reported can probably Fuchs S, Sontag G, Stidl R, Ehrilich V, Kundi M, Knasmüller S (2008)
be attributed to the use of different cell lines, different Detoxification of patulin and ochratoxin A, two abundant mycotox-
endpoints (e.g., MTT reduction, neutral red uptake, cell ins, by lactic acid bacteria. Food Chem Toxicol 43: 1398–1407. DOI:
10.1016/j.fct.2007.10.008.
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absence of serum in the culture medium (Dietrich et al., Haskard CA, El-Nezami H, Kankaanpää P, Salminen S, Ahokas J
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CONCLUSION Heussner AH, Dietrich DR, Brien EO (2006) In vitro investigation of
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Acknowledgements Knasmüller S, Steinkellner H, Hirschl AM, Rabot S, Nobis EC, Kassie
F (2001) Impact of bacteria in dairy products and of the intestinal
The study was supported by the Polish State Com- microflora on the genotoxic and carcinogenic effects of heterocy-
mittee for Research (grant R12 028 01). We would like clic aromatic amines. Mutat Res 480–481: 129–138. DOI: 10.1016/
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Antigenotoxic effect of Saccharomyces cerevisiae on the damage pro-
Weigl Conference on Microbiology, Gdańsk, 2015. duced in mice fed with aflatoxin B1 contaminated corn. Food Chem
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