BOTANICA LITHUANICA 2005, Suppl. 7: 123–129

BOTANICA LITHUANICA 2005, Suppl. 7: 123–129
USE OF BIOLOGICAL METHOD FOR DETOXIFICATION OF MYCOTOXINS
Bronius BAKUTIS 1 , Violeta BALIUKONIENË 1 , Algimantas PAÐKEVIÈIUS 2
1
Lithuanian Veterinary Academy, Tilþës Str. 18, LT-47181 Kaunas, Lithuania; e-mail violeta.baliukoniene@lva.lt
2
Institute of Botany, Laboratory of Biodeterioration Research, Þaliøjø Eþerø Str. 49, LT-08406 Vilnius,
Lithuania; e-mail a.paskevicius@botanika.lt
Abstract
Bakutis B., Baliukonienë V., Paðkevièius A., 2005: Use of biological method for detoxification of
mycotoxins [Biologinio metodo taikymas mikotoksinø detoksikacijai]. – Botanica Lithuanica, Suppl.
7: 123–129.
Mycotoxins are secondary metabolites secreted by fungi, mostly belonging to the genera Aspergillus
Mich. : Fr., Penicillium Link and Fusarium Link. Mycotoxin-containing feed can cause serious
diseases in farm animals. The presence of mycotoxins in feed may decrease feed intake and affect
animal performance. The most applied method for protecting animals against mycotoxicosis is the
utilization of adsorbents mixed with the feed, which are supposed to bind the mycotoxins efficiently
in the gastro-intestinal tract. The most promising approach to decontaminate feed is a biological
detoxification.
Keywords: adsorbents, biological detoxification, fungi, mycotoxins, yeast.
INTRODUCTION
Fungi are ubiquitous in nature and fulfill an essential
role in the recycling of nutrients from decaying matter
in soils, vegetation and water. Fungi imperfecti are known
to produce variety of secondary metabolites that seem
to improve their competitiveness in nature (STEYN, 1998).
The primary metabolites of fungi and other organisms
are those compounds that are essential for growth. Secondary
metabolites are formed in the final stages of the
exponential growth phase (JAY, 2000). Fungal metabolites
exhibit an intrinsic toxicity even at low concentrations,
resulting in their collective classification as mycotoxins
(FINK-GREMMELS & GEORGIOU, 1996). Mycotoxins
are unavoidable contaminants in foods and feeds and
are a major problem all over the world (D’MELLO et al.,
1999). The number of mycotoxins known to induce
signs of toxicity in mammalian and avian species exceeds
300 (FINK-GREMMELS, 1999) and is steadily increasing.
Mycotoxins are important because they cause undesirable
biological reactions when ingested. These reactions
may vary from acute, over disease and death, to chronic
disease states, and economically important but clinically
obscure changes in growth, production and immunosuppression.
There are thousands of secondary fungal metabolites,
the vast majority of which have not been tested
for toxicity or unequivocally associated with disease
outbreaks (BRYDEN, 2004). The most significant mycotoxins
in naturally contaminated foods and feeds are
aflatoxins, ochratoxins, trichothecenes, zearalenone and
fumonisins (DÄNICKE, 2002). In many cases these mycotoxins
can be found in combination in food (VER-
SANTVOORT et al., 2005) and feed (GARALEVIÈIENË et al.,
2003).
Fungi of the genus Fusarium are common plant pathogens
occurring world wide in a variety of crops, although
they are mainly associated with cereals. Fusarium
species can produce over one hundred secondary
123

metabolites. The most important Fusarium mycotoxins
that can frequently occur at biologically significant concentrations
in cereals, are trichothecenes (deoxynivalenol,
nivalenol and T-2 toxin), zearalenone, fumonisins
(mainly B 1
and B 2
) (RICHARD, 2003). These compounds
can occur naturally in agricultural food products, either
individually or as specific clusters of two or more of them
depending on the producing fungal species. They have
been implicated alone or in combination between them
and/or with other mycotoxins as the causative agents in a
variety of animal diseases and have been associated to
some human diseases (D’MELLO et al., 1999).
The important mycotoxins produced by Aspergillus
species include aflatoxins, ochratoxin, sterigmatocystin,
cyclopiazonic acid. Aflatoxins are produced mainly by
A. flavus, A. parasiticus and A. nominus, and are considered
very potent liver carcinogens in various animal
species and humans (ICMSF, 1996).
According to PITT & LEISTNER (1991) Penicillium species
can produce 27 different mycotoxins, with three
being the most important: ochratoxins, patulin, and citrinin.
Ochratoxin A is a potent nephrotoxin, teratogen,
and carcinogen. Patulin produces adverse neurological
and gastrointestinal effects and is produced by P. expansum.
Citrinin is a nephrotoxin and is produced mainly
by P. citrinum, P. expansum, and P. verrucosum (PITT,
1997).
Various approaches have been identified to reduce
or prevent the adverse effects of mycotoxins on animal
health and production. The most promising approach
to decontaminate feed is the biological detoxification,
the so-called biotransformation (YIANNIKOURIS & JOUA-
NY, 2002). More than twenty bacteria and yeast with mycotoxins-detoxifying
properties could be isolated and
identified (SCHATZMAYR et al., 2004).
SCHATZMAYR et al. (2004) investigated yeast of the
genera Trichosporon, Rhodotorula and Cryptococcus
for their ochratoxin A detoxifying activity. However,
all these strains were scientifically investigated for their
potential use as feed additive. At the end of a very comprehensive
selection process a new yeast species currently
registered as Trichosporon mycotoxinivorans came
out on top. A combination of selected minerals, Eubacterium
BBSH 797 strain and Trichosporon mycotoxinivorans
can be used to prevent swine from mycotoxicoses
caused by aflatoxins, trichothecenes, zearalenone
and ochratoxin A (SCHATZMAYR et al., 2004).
One product, which has been recently developed and
addresses the features above, is a natural absorbent derived
from the cell wall of Saccharomyces cerevisae.
Modified yeast cell wall mannanoligosaccharide (MOS)
has been reported to effectively bind aflatoxins, and to
bind ochratoxins and the fusariotoxins to a lesser degree
(DEVEGOWDA et al., 1998).
The aim of this article is to present detoxifiing adsorbents
for mycotoxins. In addition, we also include our
results obtained during scientific studies of different
yeast influence on mycotoxins in the feed.
MATERIAL AND METHODS
Samples of sunflower seed cake, maize, maize fodder,
composed fodder were selected for the primary investigation.
Fodder samples were moistened with produced
medium (6.7 g/l) (Difco, Yeast nitrogen base, USA).
Fodder was inoculated with Saccharomycopsis capsularis
Schiöonning, Saccharomyces cerevisiae Hansen,
Candida utilis (Henneberg) Kreger-van Rij, Geotrichum
fermentans (Didens et Lodder) Arx, Rhodotorula rubra
(Demme) Lodder, Rhodotorula glutinis (Fresenius) Harrison,
Metschnikowia pulcherima Pitt et Miller, Kluyveromyces
marxianus (Hansen) van der Walt yeast species.
Inoculant concentration was 10 4 cell/ml. Yeast was cultivated
for 10 days in a thermostat at a temperature of 28
± 2 o C.
Mycotoxin analysis was carried out before and after
the investigation. Fodder samples were analysed by the
Elisa (enzyme-linked immunosorbent assay) method
(CHU, 1996). The Veratox ® Don 5/5 (Neogen, USA),
Veratox ® T-2 toxin (Neogen, USA), Veratox ® Zearalenone
(Neogen, USA), Veratox ® Ochratoxin (Neogen,
USA), Veratox ® Aflatoxin (Neogen, USA) were used for
the analysis. Mycotoxin extraction and tests were performed
according to the manufacturer’s instruction. The
obtained data were processed using Microsoft Excel XP.
RESULTS AND DISCUSSION
Recent progress in yeast biotechnology and carbohydrate
chemistry has opened new avenues to highlight
mycotoxin problems. The use of biological method to
detoxify mycotoxins is based on elimination of the toxin
(adsorption), elimination of the toxicity (biotransformation)
and elimination of toxin-related effects (BAKUTIS,
2004).
In 1993, researchers supplemented an aflatoxin-contaminated
broiler diet with 0.2 % live yeast (Yea-Sacc ®
1026) and reported significant improvement in the birds’
weight gain and feed efficiency (STANLEY et al., 1993).
Researchers in different countries have reported beneficial
effects of the inner cell wall fraction of yeast
across a wide range of mycotoxins based on in vitro
and in vivo tests in poultry (MANOJ & DEVEGOWDA, 2000),
124

Table 1.
Capacity of glucomannans from Saccharomyces cerevisae
to adsorb mycotoxins (adapted from YIANNIKOURIS &
JOUANY, 2002)
Mycotoxins Adsorbing (%)
Aflatoxins (total) 95.0
Fumonisins 67.0
Zearalenone 77.0
T-2 toxin 33.4
Citrinin 18.4
Deoxynivalenol 12.6
Ochratoxin A 12.5
Nivalenol 8.2
pigs (SWAMY et al., 2003), and dairy (WHITLOW et al.,
2000).
Fermentation by Saccharomyces cerevisae of wortcontaining
zearalenone results in conversion of 69 %
of toxin to beta-zearalenol, a metabolite of lower activity
than the parent compound (KARLOVSKY, 1999).
Glucomannans extracted from the external part of
cell wall of the yeast Saccharomyces cerevisae are able
to bind certain mycotoxins (Table 1). Their great binding
capacity results from the large area available for exchange.
Thus, 500 g of glucomanns from yeast cell-wall have
the same adsorption capacity as 8 kg of clay (YIANNI-
KOURIS & JOUANY, 2002).
A feeding trial conducted with broiler chickens revealed
that the negative influence of high doses of ochratoxin
A on the performance of broilers could be neutralized
by addition of stabilized Trichosporon spp. cells.
The final weight of the group receiving 1 ppm (mg/kg)
ochratoxin A and yeast (10 5 CFU per gram feed) was on
average by 61 gram higher than the positive control
group (1 ppm ochratoxin A without additive) (SCHATZ-
MAYR et al., 2004).
Incubation experiments with new yeast strain Trichosporon
mycotoxinivorans showed that zearalenone
could also be successfully degraded. Tests with animal
cell cultures carried out at Utrecht University (the Netherlands)
have already shown that samples, incubated
with the yeast strain and zearalenone for a certain period
of time did no longer show estrogenic effects (SCHAT-
ZMAYR et al., 2004).
Now various mycotoxin-detoxifying adsorbents are
suggested in Lithuania. The mycotoxin adsorbents with
yeast, yeast cell wall constituents are presented on Table 2.
Many adsorbents were tested for binding of several
mycotoxins both in vitro and in vivo. The degree of ad-
Table 2.
Mycotoxin adsorbents with yeast
Name of adsorbent
Toxy-nil dry
Sorbic acid, citric acid; calcium
propionate; copper sulphate;
Inactivated yeast
(Saccharomyces cerevisiae)
Mycofix plus 3.0
Inactivated yeast (specific types
of Saccharomyces),
diatomaceous earth (E551 c),
caolinite clay, free of asbestus
(E559)
Mycosorb
Brewers dried yeast; dried
Saccharomyces cerevisiae
fermentation solubles
Bio-mos
Brewers dried yeast; dried
Saccharomyces cerevisiae
fermentation solubles
Toxy-nil plus dry
Copper sulphate; HSCAS
(sepiolite, bentonite); dried
yeast (Saccharomyces
cerevisiae)
Klinosan
Clinoptilolite; yeast extract
(Saccharomyces cerevisiae)
Manufacturer
Nutri-Ad
International N.V.,
Belgium
Biomin Gesunde
Tierernahrung
International,
Austria
Alltech Hungary
Kft., Hungary
Alltech Hungary
Kft., Hungary
Nutri-Ad
International N.V.,
Belgium
Unipoint AG,
Switzerland
sorption in vitro depends on the chemical nature of the
mycotoxin in relation to the surface properties and the
geometry of the adsorbent. If the mycotoxin is bound
strongly enough to the adsorbent, its adsorption by the
digestive tract will be hindered (SCHATZMAYR et al., 2004).
Often mycotoxin concentrations range between 10
to 100 ppb in the feed. Whilst these may not cause clinical
symptoms in animals, they do, nevertheless, reduce
animal performance through the effects on feed intake,
weight gain, immune status and reproduction. In such
situations the adsorbents should have a very high affinity
for mycotoxins, so that even very low concentrations can
be adsorbed (SWAMY et al., 2003).
The results of present study showed that selected various
yeast species can reduce the different mycotoxins,
when yeast is cultivated in the various plant substrates.
The cultivation of Rhodotorula rubra yeast reduced the
amount of deoxynivalenol from 0.35 to 0.19 mg/kg in
the sunflower seed cake in 10 days. That came to 47.7 %.
125

Mycotoxin amount (mg/kg)
2,5
2,0
1,5
1,0
0,5
0,0
2
0,35
0,0059 0
0,19
0
AFL DON ZON
Mycotoxin
Fig. 1.
The amount of mycotoxins (mg/kg) in the sunflower seed cake before and after 10 days cultivation of
Rhodotorula rubra yeasts: AFL – aflatoxins, DON – deoxynivalenol, ZON – zearalenone
Mycotoxin amount (mg/kg)
2,5
2,0
1,5
1,0
0,5
0,0
2,2
0,3 0,222
0,015 0,0047 0,0059
DON ZON OA
Mycotoxin
Fig. 2.
The amount of mycotoxins (mg/kg) in the maize fodder before and after 10 days cultivation of Rhodotorula
glutinis yeasts: OA – ochratoxins, DON – deoxynivalenol, ZON – zearalenone
The amounts of aflatoxins and zearalenone disappeared
(Fig. 1).
Rhodotorula glutinis yeast reduced the amount of
deoxynivalenol from 2.20 to 0.30 mg/kg, the amount of
zearalenone – from 0.222 to 0.015 mg/kg in the maize
fodder in 10 days. The amount of deoxynivalenol was
reduced by 84.6 % and zearalenone – 93.2 %. This yeast
species increased the amount of ochratoxins from
0.0047 to 0.0059 mg/kg in the maize fodder in 10 days.
That made 20.3 % (Fig. 2).
Metschnikowia pulcherima yeast reduced the amount
of deoxynivalenol from 0.20 to 0.09 mg/kg in the maize
in 10 days. That came to 55% (Fig. 3).
Geotrichum fermentans yeast reduced the amount
of deoxynivalenol from 0.75 to 0.28 mg/kg, the amount
of zearalenone – from 0.20 to 0.11 mg/kg in the compo-
126

0,3
0,2
Mycotoxin amount (mg/kg)
0,2
0,2
0,1
0,1
0,09
0,0
DON
Mycotoxin
Fig. 3.
The amount of deoxynivalenol (mg/kg) in the maize before and after 10 days cultivation of Metschnikowia
pulcherrima yeasts
0,8
0,75
0,7
Mycotoxin amount (mg/kg)
0,6
0,5
0,4
0,3
0,2
0,1
0,0
0,01
0
0,28
0,2
0,11
AFL DON ZON
Mycotoxin
Fig. 4.
The amount of mycotoxins (mg/kg) in the composed fodder before and after 10 days cultivation of Geotrichum
fermentans yeasts: AFL – aflatoxins, DON – deoxynivalenol, ZON – zearalenone
sed fodder in 10 days. The amount of deoxynivalenol
was reduced by 62.7 % and zearalenone – 45.0 %.
Aflatoxins disappeared (Fig. 4).
The cultivation of Kluyveromyces marxianus yeast
reduced the amount of deoxynivalenol from 0.25 to 0.14
mg/kg in the composed fodder in 10 days. The amount
of deoxynivalenol was reduced by 44 %. The amount of
aflatoxins was lower and came to 0.0045 mg/kg in the
composed fodder, this amount of aflatoxins disappeared
(Fig. 5).
CONCLUSIONS
Biological methods are a relatively new approach
to the decontamination of toxic feeds that already proved
to work. They decontaminate the mycotoxins via struc-
127

Mycotoxin amount (mg/kg)
0,8
0,7
0,6
0,5
0,4
0,3
0,2
0,1
0
0,7
0,25
0,18 0,14
0,0045 0
AFL DON ZON
Mycotoxin
Fig. 5.
The amount of mycotoxins (mg/kg) in the composed fodder before and after 10 days cultivation of Kluyveromyces
marxianus yeasts: AFL – aflatoxins, DON – deoxynivalenol, ZON – zearalenone
tural changes in the mycotoxin molecule without the
generation of any toxic by-products or metabolites.
Furthermore, this treatment does not have any impact
on the nutrient value, availability of vitamins and trace
elements and the palatability is not influenced.
The isolation and characterization of microorganisms
(yeasts) that are able to bio-transform mycotoxins
is probably the breakthrough for the practical application
of biotechnology in respective decontamination
processes taking place directly in the intestinal tract of
animals. Biological methods described above may
become the technology of choice, as enzymatic reactions
offer a specific, irreversible, efficient and environmentally
friendly way of detoxification that leaves neither
toxic residues nor any undesired by-products.
In this study, the detoxifying yeast species were selected.
The cultivation of Rhodotorula rubra yeast reduced
deoxynivalenol by 47.7 %, zearalenone and aflatoxins
– 100 % in the sunflower seed cake. In the maize
fodder, Rhodotorula glutinis yeast reduced deoxynivalenol
by 84.6 % and zearalenone by 93.2 %. Metschnikowia
pulcherima yeast reduced deoxynivalenol by 55 %
in the maize. Geotrichum fermentans yeast reduced deoxynivalenol
by 62.7 %, zearalenone – 45.0 % and aflatoxins
– 100 % in the composed fodder. Kluyveromyces
marxianus yeast reduced deoxynivalenol by 44 %, aflatoxins
– 100 % in the composed fodder.
The results of this study require further investigations.
ACKNOWLEDGEMENTS
This study was supported by the Lithuanian State
Science and Studies Foundation.
REFERENCES
BAKUTIS B., 2004: Mikotoksinai gyvuliø paðaruose. –
Kaunas.
BRYDEN W. L., 2004: Acute and chronic toxicology of
mycotoxins in animal production. – World Nutrition
Forum 2004: 25–26. – Erber.
RICHARD J. L., 2003: Mycotoxins: Risk in Plant, Animal
and Human Systems. – Ames.
CHU F. S., 1996: Recent studies on immunoassays for
mycotoxins. – In: BEIER R. C., STANKER L. H. (eds.),
Immunoassays for residue analysis. – Food Safety.
American Chemical Society: 294–313. – Washington.
DÄNICKE S., 2002: Prevention and control of mycotoxins.
– World’s Poultry Science, 58: 452–475.
DEVEGOWDA G., RADU M. V. L. N., NAZAR A., SWAMY H.
V. L. M., 1998: Mycotoxin picture worldwide: Novel
solutions for their counteraction. – Proceedings of
Alltech’s 14th Annual Symposium: 241–255.
D’MELLO J. P. F., PLACINTA C. M., MCDONALD A. C. M.,
1999: Fusarium mycotoxins: a review of global implications
for animal health, welfare and productivity.
– Animal Feed Science and Technology, 80:
183–205.
128

FINK-GREMMELS J., GEORGIOU N. A., 1996: Risk assessment
of mycotoxins for the consumer. – In: ENNEN
G., KUIPER H. A., VALENTIN A. (eds.), Residues of
Veterinary Drugs and Mycotoxins in Animal Products:
159–174. – Wageningen.
FINK-GREMMELS J., 1999: Mycotoxins: their implications
for human and animal health. – Veterinary Quarterly,
21: 115–120.
GARALEVIÈIENË D., PETTERSSON H., AGNEDAL M., 2003:
Occurrence of trichothecenes, zearalenone and ochratoxin
A in cereals and mixed feed from central
Lithuania. – Mycotoxin Research, 18: 77–89.
ICMSF (International Commission on Microbiological
Specification for Food), 1996: Toxigenic fungi: Aspergillus.
– In: Microorganisms in Food 5: Characteristics
of Microbial Pathogens: 347–381. – London.
JAY J. M., 2000: Modern Food Microbiology. – Maryland.
KARLOVSKY P., 1999: Biological detoxification of fungal
toxins and its use in plant breeding, feed and food
production. – Natural Toxins, 7: 1–23.
MANOJ K. B., DEVEGOWDA G., 2000: Efficacy of esterified
glucomannan to ameliorate the toxic effects
of T-2 toxin in layer hens – an in vitro study. – Proceedings
CC Worlds Poultry Congress. New Delhi,
India, 4: 296.
PITT J. I., LEISTNER L., 1991: Toxigenic Penicillium species.
– In: SMITH J. E., HENDERSON R. S. (eds.), Mycotoxins
and Animal Food: 91–99. – Boca Raton.
PITT J. L., 1997: Toxigenic Penicillium spiecies. – In:
DOYLE M. P., BEUCHAT L. R., MONTVILLE T. J. (eds.),
Food Microbiology, Fundamentals and Frontiers:
406–418. – Washington.
SCHATZMAYR D., NITSCH S., BINDER E. M., TÄUBEL M.,
LOIBNER A., SCHATZMAYR G., 2004: Detoxification
of mycotoxins. – World Nutrition Forum 2004: 128–
129. – Erber.
STANLEY V. G., OJO S., WOLDESENBET S., HUTCHINSON D.
H., KUBENA L. F., 1993: The use of Saccharomyces
cerevisiae to suppress the effects of aflatoxicosis in
broiler chicks. – Poultry Science, 72: 1867–1872.
STEYN P. S., 1998: The biosynthesis of mycotoxins. –
Revue de Medicine Veterinaire, 149: 469–478.
SWAMY H. V., SMITH T. K., MACDONALD E. J., KARROW
N. A., WOODWARD B., BOERMANS H. J., 2003: Effects
of feeding a blend of grains naturally contaminated
with Fusarium mycotoxins on growth and immunological
measurements of starter pigs, and the efficacy
of a polymeric glucomannan mycotoxin adsorbent. –
Animal Science, 81: 2792–2803.
VERSANTVOORT C. H., OOMEN A. G., VAN DE KAMP E.,
ROMPELBERG C. J., SIPS A. J., 2005: Applicability of
an in vitro digestion model in assessing the bioaccessibility
of mycotoxins from food. – Food Chemical
Toxicology, 43(1): 31–40.
WHITLOW L., DIAZ D. E., HOPKINS B. A., HAGLER W. M.,
2000: Mycotoxins and milk safety: the potential to
block transfer to milk. – In: LYONS T. P., JACQUES K.
A. (eds.), Biotechnology in the Feed Industry: 391–
408. – Loughborough Lakes.
YIANNIKOURIS A., JOUANY J., 2002: Mycotoxins in feed
and their fate in animals: a review. – Animal Research,
51: 81–99.
BIOLOGINIO METODO TAIKYMAS MIKOTOKSINØ DETOKSIKACIJAI
Bronius BAKUTIS, Violeta BALIUKONIENË, Algimantas PAÐKEVIÈIUS
Santrauka
Mikotoksinai yra antriniai mikromicetø metabolitai.
Pagrindinius mikotoksinus gamina Aspergillus Mich. : Fr.,
Penicillium Link ir Fusarium Link genèiø mikromicetai.
Mikotoksinais uþterðti paðarai gali sukelti pavojingø ligø
gyvuliams ir paukðèiams. Mikotoksinø kiekis paðaruose
trukdo ásisavinti juose esanèias maisto medþiagas ir
sumaþina gyvuliø produktyvumà. Daþniausiai prieð
mikotoksikozes naudojami adsorbentai, kuriø maiðoma
á paðarus. Adsorbentai gyvuliø virðkinimo trakte suriða
arba suskaldo mikotoksinus. Veiksmingas bûdas detoksikuoti
paðarus yra biologinë detoksikacija. Straipsnyje
apþvelgtas ávairiø mieliø padermiø naudojimas mikotoksinø
detoksikacijai.
129

130