3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P14 TESTING OF DIFFERENT sACHArOMyCes
SPECIES FOR ThE AbILITy TO SORb
DEOxyNIVALENOL
RADKA BURDYCHOVá, VLASTIMIL DOHnAL and
DAnA VRánOVá
Mendel University of Agriculture and Forestry in Brno
Zemedelska 1, 613 00 Brno, Czech Republic,
burdycho@node.mendelu.cz
Introduction
Mycotoxin contamination of food and feed is a high
risk to human and animal health 2 . The majority of the causal
organisms are producers of mycotoxins such as highly toxic
trichothecenes 4 . Four types of trichothecenes are described,
type A and type B, which differ in the presence or absence of
a keto group at C-8 of the trichothecene skeleton, type C with
additional epoxydic group and macrocyclic type D. The most
common trichothecene in cereals is type B trichothecene deoxynivalenol
(DOn).
Deoxynivalenol, also called vomitoxin, is produced by
Fusarium fungi, such as F. graminearum or F. culmorum. It
has negative effect on animal growth and health. DOn inhibits
the synthesis of DnA, RnA and proteins at the ribosomal
level. High doses causes the vomiting in pigs, lower
concentrations in the diet reduces feed intake and animal
growth 5 . Different physical and chemical methods have been
recommended for detoxification of mycotoxin-contaminated
food and feed. nevertheless, only a few of them have been
accepted for practical use. Thermal degradation of trichothecenes
is not so effective, because they are relatively stable
and they decomposes at 210 °C within 30–40 min 10 . From
physical methods are the most frequently used feed additives
on sorbent basis, such as bentonite or active charcoil. The
disadvantages of adsorbents are their relatively high dosage
and sorption of biologically active compounds, e.g. vitamins
or trace metals. In addition, the sorbents can bind to
only a limited group of mycotoxins and in some cases does
not provide required effect 7 . Biological decontamination of
mycotoxins using microorganisms is one of the well-known
strategies for the management of mycotoxins in food and
feed. Biological decontamination of mycotoxins by different
microorganisms was reviewed several times. 1,8,11,15 There
are two ways of action – sorption on cell walls or enzymatic
degradation, for example using epoxydase. De-epoxylated
form is less toxic than original DOn. Among the different
potential decontaminating microorganisms, the genus Saccharomyces
represent the unique group, which is widely used
in food fermentation and preservation. The aim of this study
was screening of ability of different Saccharomyces species
to remove deoxynivalenol from liquid medium.
s605
Experimental
M i c r o b i a l C u l t u r e s a n d C u l t u r e
C o n d i t i o n s
All cultures were obtained from Culture Collection of
Yeast (Bratislava, Slovakia). The cultures were Saccharomyces
cerevisiae 20 1 ; isolated from loaf of hornbeam, Saccharomyces
bayanus 21-31-12; isolated from mushrooms,
Saccharomyces paradoxus 2 isolated from needles of spruce,
Saccharomyces paradoxus 21-53-2 isolated from soil and
Saccharomyces paradoxus isolated from the loaf of locust.
All cultures were cultivated in Plate Count Broth (PCB;
Merck, Germany) at 30 °C for 48 hours and then subcultured
by transfering 4 ml of the culture to the cultivation test tube.
Three replicates per sample of inoculum were used at each
measuring. On the base of O. D. (600 nm) values that have
been taken during the cultivation of yeasts with deoxinyvalenol
the analysis of their counts has been done.
P r e p a r a t i o n o f Y e a s t C u l t u r e s f o r
T e s t i n g o f D O n S o r b t i o n
All used chemicals were analytical or gradient grade.
Standard of deoxynivalenol (DOn) was obtained from
Sigma-Aldrich, s.r.o. (Czech Republic). The stock solution
of DOn was prepared by dissolution of 1 mg of DOn
in 5 ml of acetonitrile to give solution with concentration
0.2 mg ml –1 . The solution was stored at –18 °C. Working
solutions for calibration curve measurement were prepared
by dilution of stock solution.
The amount of 0.5 μg of DOn was transferred to test
tube and evaporated to dryness. In the next step, 4 ml of cultivation
media (PCB) with yeasts were added to the test tube
and the sample was cultivated in thermostated box at 30 °C
for 4 hours. The concentration of free DOn was measured at
the beginning of cultivation and after 4 hours of cultivation.
Prior the DOn determination it was necessary to remove
yeasts from culture medium using ultrafiltration through
polytetrafluoroethylene membrane filter (SMI-LabHut Ltd.,
UK) with pore size 0.20 μm. After this step, the filtrate was
diluted with acetonitrile (in ratio 16 : 84). next, the clean-up
with MycoSep ® 225 Trich was applied. Briefly, 5 ml of solution
was transferred to the glass tube and pushed through the
MycoSep ® 225 Trich column. 2 ml of this eluate were evaporated
to dryness and redissolved in 400 μl of HPLC mobile
phase. Mobile phase consisted of 1mM formic acid/acetonitrile
(90 : 10, v:v) with flow rate 1 ml min –1 .
C h r o m a t o g r a p h i c D e t e r m i n a t i o n o f
D O n
The HPLC system HP 1100 (Agilent Technologies,
Palo Alto, USA) consisted of vacuum degasser unit
(model G1322A), quaternary pump (G1311A), autosampler
(G1313A) and quadrupole mass spectrometer (G1946VL)
with electrospray ionization was used. The ChemStation software
(Rev. A 10.02) controllig chromatographic system and
was used for chromatogram evaluation.