Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing

Toxin production by bacterial endosymbionts of a Rhizopus microsporus strain used for tempe/sufu processing

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Short Communication
Toxin production by bacterial endosymbionts of a
Rhizopus microsporus
strain used
for tempe/sufu processing
Barbara Rohm
a
, Kirstin Scherlach
a
, Nadine Möbius
a
, Laila P. Partida-Martinez
a
,
1
, Christian Hertweck
a
,
b
,
?
a
Leibniz Institute for Natural Product Research and Infection Biology (HKI), Dept. of Biomolecular Chemistry, Beutenbergstr. 11a, 07745 Jena, Germ
any
b
Friedrich Schiller University, Jena, Germany
abstract
article  info
Article history:
Received 3 April 2009
Received in revised form 1 October 2009
Accepted 12 October 2009
Keywords:
Mycotoxin
Rhizopus
Burkholderia
Rhizoxin
Sufu
Tempe
Mould fungi are not only well known for food spoilage through toxin formation but also for the production of
fermented foods. In Asian countries, the fermentation of soy beans and tofu for tempe and sufu production
with various
Rhizopus
strains is widespread. Here we report the
fi
nding of toxinogenic bacteria in a starter
culture used for sufu production. By means of metabolic pro
fi
ling of the fungus under standard conditions for
tempe and sufu production, we found that toxins of the rhizoxin complex are produced in critical amounts.
Considering that rhizoxins are severe toxins with strong antimitotic activity it is important to notice that our
fi
ndings uncover a health-threatening symbiosis in food processing. A simple PCR method for detecting
toxinogenic endofungal bacteria in starter cultures is proposed.
© 2009 Elsevier B.V. All rights reserved.
1. Introduction
Mould (
fi
lamentous) fungi are infamous for causing spoilage of food
and production of toxic metabolites (
Bohra and Purohit, 2003; Richard,
2007
). Fungal toxins not only cause massive losses in the food industry,
but

more importantly

threaten the health of consumers (
Hussein
and Brasel, 2001
). Mass intoxi
fi
cations of animals and humans such as
the epidemic Turkey

X

disease and the historical and modern cases of
ergotism (St. Antony’s Fire) are infamous examples of mycotoxicoses,
underscoring the global impact of food spoilage by fungi (
Franck, 1984;
Möbius and Hertweck, 2009
). Furthermore, even low concentrations of
mycotoxinsin food canelicit deleterious effects,suchaschronic or acute
toxic damage of liver and kidney (
Bohra and Purohit, 2003; Fung and
Clark, 2004
). On the other hand, mould fungi also play a key role as
sources of enzymes in the re
fi
nement of agricultural goods, such as
cheese (e.g.
Penicillium
spp.) (
Weidenbörner, 2008
). In Asian countries,
the use of Zygomycetes in the fermentation of soy beans to produce
tempe and sufu (

soy bean cheese

)ismorecommon(
Han et al., 2001
).
However, it is evident that microbial food fermentations may also be
hazardousduetotheusageofwrongmediaorunsuitable (i.e. toxigenic)
strains, and/or possible microbial contaminations (
Daniel and Fung,
2000
). In the case of
Rhizopus
spp., two types of mycotoxins, the
rhizoxins and the rhizonins, are known that were implicated as
potential food contaminants (
Jennessen et al., 2005
).
Recently,we have discovered the
fi
rst cases in whichmycotoxinsare
actually not biosynthesized by the fungus, but by

endofungal

bacteria
residing within the cytosol (
Partida-Martinez and Hertweck, 2005;
Partida-Martinez et al., 2007a; Lackner et al., 2009
). We found that
various
Rhizopus microsporus
strains harbour bacterial symbionts such
as
Burkholderia rhizoxinica
and
Burkholderia endofungorum
(
Partida-
Martinez et al., 2007c
), which produce the antimitotic polyketide
macrolide rhizoxin (
Partida-Martinez and Hertweck, 2007; Scherlach
et al., 2006
), and the hepatotoxic cyclopeptide rhizonin (
Partida-
Martinez et al., 2007b
), respectively. Rhizoxin has been shown to be
toxic to a variety of human and murine tumor cell lines (
Tsuruo et al.,
1986
). It effectively inhibits cell division by preventing the assembly of
microtubules in eukaryotic cells (
Takahashi et al., 1987
). Rhizonin
causes severe hepatic lesions and leads to 100% mortality in rats due to
acute and chronic failure of the liver (
Wilson et al., 1984
).
Here we report the
fi
nding of a related toxinogenic symbiosis in a
starter culture for sufu fermentation. We demonstrate that these
antimitotic toxins are produced even under the standard conditions
employed for soy bean fermentation.
2. Materials and methods
2.1. Detection and isolation of bacterial endosymbionts
R. microsporus
var.
microsporus
Tieghem (CBS 111563) was
cultivated at 30 °C and 80 rpm in VK medium composed of 1% corn
International Journal of Food Microbiology 136 (2010) 368

371
?
Corresponding author. Leibniz Institute for Natural Product Research and Infection
Biology (HKI), Dept. of Biomolecular Chemistry, Beutenbergstr. 11a, 07745 Jena,
Germany. Tel.: +49 3641 5321100; fax: +49 3641 5320804.
E-mail address:
[email protected]
(C. Hertweck).
1
Current address: Department of Biotechnology and Food Engineering, Instituto
Tecnológico y de Estudios Superiores de Monterrey (ITESM), Monterrey, Nuevo León
64849, Mexico.
0168-1605/$

see front matter © 2009 Elsevier B.V. All rights reserved.
doi:
10.1016/j.ijfoodmicro.2009.10.010
Contents lists available at
ScienceDirect
International Journal of Food Microbiology
journal homepage: www.elsevier.com/locate/ijfoodmicro
starch, 0.5% glycerol, 1% gluten meal, 1% dried yeast, 1% corn steep
liquor, and 1% CaCO
3
, pH=6.5. After 4 days, a small mycelial pellet
(0.5 mL) was aseptically taken and submerged in 500
µ
L 0.85% NaCl.
Using mechanical stress (pipetting), the mycelium was broken and
then submitted to centrifugation (30 min, 13,200 rpm). A loop of the
supernatant was then plated on nutrient agar (Becton, Dickinson and
Company, USA) plates. The plates were incubated at 30 °C for several
days, until the presence of mycelial or bacterial colonies could be
con
fi
rmed. Once the
fi
rst bacterial colonies appeared, they were
picked up and cultivated in 1 mL of tryptic soy broth (TSB, Merck,
Darmstadt, Germany) at 30 °C and 150

200 rpm until growth could
be seen, usually after 2

3 days. Analysis of 16S rRNA genes and
microscopy was performed to identify the isolates as
Burkholderia
sp.
(
Partida-Martinez et al., 2007a; Lackner et al., 2009
). For generation of
symbiont-free
R. microsporus
, the strain was constantly cultivated in
the presence of cipro
fl
oxacin (20

40
µ
gmL

1
, Bayer AG) (
Partida-
Martinez et al., 2007a
).
2.2. Laser microscopy
A sterile cover slip was placed into a potato dextrose agar (PDA)
plate, which was previously inoculated with
R. microsporus
var.
mi-
crosporus
Tieghem. The cover slip remained in the plate until several
hyphae grew on it. The mycelium on the cover slip was incubated in
the dark for 15 min with 0.025 µL of SYTO 9 green-
fl
uorescent nucleic
acid stain and an equal amount of propidium iodide stain dissolved in
50 µL trypton soy bouillon. Microscopic analysis was performed using
a Zeiss LSM 510 Meta Laser Microscope at 480/500 nm (
Partida-
Martinez and Hertweck, 2005
).
2.3. DNA isolation and PCR
Metagenomic DNA from fungal species was obtained using
MasterPure

Yeast DNA puri
fi
cation kit (Epicentre Biotechnologies)
with several modi
fi
cations for fungal strains (
Schmitt et al., 2008;
Partida-Martinez et al., 2008; Lackner et al., 2009
). Mycelium obtained
from a 3 d grown culture was separated from the media and after
rinsing with water dried with a miracloth paper. The mycelium was
ground with glass beads, mixed with 450 µL Yeast Cell Lysis solution
and incubated for 60 min at 65 °C under heavy shaking. After 5 min on
ice, 225 µL MPC protein precipitation solution (Epicentre Biotechnol-
ogies) were added and centrifuged at 10,000 rpm for 10 min. The DNA
in the supernatant was precipitated with 750 µL isopropanol and
pelleted by centrifugation. After washing with 70% ethanol, the pellet
was dissolved in 50 µL TE buffer.
Bacterial DNA was obtained by chloroform/phenol extraction after
3 d incubation of the isolated bacteria in TSB at 30 °C. Bacterial cells
were lysed using a sucrose containing buffer, lysozyme and proteinase
K. After incubation with RNase A two rounds of phenol/chloroform
extraction followed. The DNA was precipitated with isopropanol,
washed with 70% ethanol and dissolved in water.
Ampli
fi
cation of polyketide synthase (PKS) genes in extracts of
fungal and bacterial DNA was performed using degenerated KS
(ketosynthase) primers as described previously (
Brendel et al., 2007;
Partida-Martinez and Hertweck, 2007
).
2.4. Fermentation and metabolic pro
fi
ling
A. Sufu. 26 g of 2 cm sized commercially supplied tofu chunks were
fi
lled into a 300 mL Erlenmeyer
fl
ask. To each
fl
ask 2 mL 0.8% citric
acid solution and 2 mL 2% NaCl solution were added. The
fl
asks were
cappedwith cotton wooland sterilized at100 °Cfor15 min.The tofu
chunks were inoculated with a small amount of mycelium of
R. mi-
crosporus
var.
microsporus
Tieghem obtained from a 3 d culture
grown on a potato-dextrose agar plate. Fermentation of the tofu was
accomplished at 23 °C for 24 h to 7 d (
Weidenbörner, 2008
).
B. Tempe. 500 g of dried soy beans were soaked in water overnight
and afterwards cooked by boiling for 1 h. After peeling of the hull,
80 g soy beans were
fi
lled in a polypropylene bag and inoculated
with
R. microsporus
var.
microsporus
Tieghem as described above.
The polypropylene bag was perforated to assure oxygen supply
and incubated at 30 °C for 24 h to 96 h (
Nout and Kiers, 2005
).
The fermented sufu/tempe was chopped into small pieces and
extracted with 150 mL ethyl acetate for 24 h. The extract was dried
with sodium sulfate and concentrated under reduced pressure. 200 µL
of the fatty residue were dissolved in 500 µL methanol. Toxin
production was monitored by HPLC analysis. Analytical HPLC was
performed on a Shimadzu HPLC system consisting of an autosampler,
high-pressure pumps, column oven and a diode array detector (DAD).
HPLC conditions (
Scherlach et al., 2006
): C18 column (Grom Sil 100
ODS 0AB, 3 µm, 250×4.6 mm) and gradient elution (MeCN/0.1% TFA
(H
2
O) 25/75 5 min, in 35 min to MeCN/0.1% TFA (H
2
O) 80/20, in 5 min
to 100% MeCN), 25 °C,
fl
ow rate 0.9 mL min

1
, injection volume
20 µL. LC-MS measurements were performed using a Surveyor HPLC
system (Thermo Electron, Bremen) coupled to a Finnigan LCQ
benchtop mass spectrometer with an electrospray ion source. HPLC
conditions: gradient elution (MeCN/0.1% HCOOH 25/75 5 min, in
35 min to MeCN/0.1% HCOOH 80/20, in 5 min to 98% MeCN);
fl
ow rate
0.6 mL min

1
). All solvents used were of analytical or HPLC grade.
Toxin concentration was measured by integration of peak areas of
rhizoxin derivatives at 311 nm and calculated in relation to the peak
area of rhizoxin S2 authentic standard (
Scherlach et al., 2006
).
3. Results and discussion
Zygomycetes, in particular
Rhizopus
spp., are commonly used for
the production of fermented soy foods such as tempe and sufu pehtze
(
Han et al., 2001, 2004
). Prompted by our discovery of a toxinogenic
Rhizopus

Burkholderia
symbiosis in the context of rice seedling blight,
we have screened a collection of
Rhizopus
spp. representing highly
diverse ecological niches and geographic origins for the occurrences of
associated bacteria (
Lackner et al., 2009
). The fungal collection also
included a
R. microsporus
var.
microsporus
Tieghem (CBS 111563)
from a

rice wine tablet

used as a sufu fermentation starter culture.
PCR using universal primers (16S rRNA genes) and total DNA obtained
from this strain indicated the presence of bacteria. Sequencing and
taxonomic classi
fi
cation revealed that these microorganisms were
related to the previously identi
fi
ed toxin-producing bacteria
B. rhi-
zoxinica
and
B. endofungorum
(
Partida-Martinez et al., 2007c
). The
rod-shaped endofungal bacteria were visualized inside the mycelium
by confocal laser scanning microscopy using a live/dead stain (
Fig. 1
).
Toxins produced under standardized fermentation conditions for both
the isolated symbionts and the fungus harbouring endobacteria were
monitored by HPLC-DAD/MS. In both cases, we noted the production
of signi
fi
cant amounts of rhizoxin derivatives 1

5. The structures of
the metabolites were deduced from retention time, UV and MS in
comparison with reference compounds (
Scherlach et al., 2006
). These
results unequivocally prove that the fungal strain used as sufu starter
culture harbours toxinogenic endobacteria and thus has, in principle,
the potential to secrete toxins into the soy bean fermentation.
However, it is well known that the production of microbial
secondary metabolites strongly depends on the fermentation condi-
tions applied and the media used (
Scherlach and Hertweck, 2009
). In
the context of food processing, a famous example is the terpenoid PR-
toxin from various strains of
Penicillium roqueforti
, which is used in
cheese re
fi
nement. However, the toxin is not produced and/or rapidly
inactivated under the conditions employed for cheese production
(
Bullerman and Bianchini, 2007; Sieber, 1978
). Likewise, roquefortine
from
P. roqueforti
and cyclopiazonic acid from
Penicillium camemberti
are not detected in harmful amounts in blue mould cheese and
camembert cheese (
Kokkonen et al., 2005; Burdock and Flamm,
369
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371