3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P55 OCCuRRENCE OF FuSARIuM MyCOTOxINS
AND ThEIR CONjuGATED FORMS IN
CEREAL-bASED FOODS
M. KOSTELAnSKá, J. HAJŠLOVá, M.
ZACHARIáŠOVá, A. KRPLOVá, J. POUSTKA and
J. KOHOUTKOVá
Institute of Chemical Technology Prague,
Technická 3, 166 28 Prague 6 – Dejvice, Czech Republic,
marta.kostelanska@vscht.cz
Introduction
Cereal-based foodstuff such as bread and beer belong
in many countries to the most important items in markets´
basket. Trichothecene mycotoxins, the toxic secondary
metabolites produced by species of Fusarium genus, are the
common “natural” contaminats of cereals worldwide. Furthermore,
these toxins are relatively stable, surviving household/industrial
processing, thus transferred into the final
food products 1,2,3 .
Deoxynivalenol (DOn) is one of the most abundant
Fusarium mycotoxin. Recently, it has been shown, that besides
the native form, DOn also occures in cereals conjugated
to glucose 1 . This “masked” DOn-3-Glucoside (DOn-3-Glc)
is produced by metabolism of living plants and therefore, the
consumers are exposed to both these mycotoxins that occur
in food. The recent studies have shown that, alike the DOn,
“masked” forms can be transmitted thoughout the processing
to food products 3 . With regard to the above mentioned, the
special attention should be paid to control maximum limits
established for toxins in unprocessed grains as well as food
products.
Several studies concern with mycotoxin contamination
of cereal-based commodities such as bread or beer, have been
published up to now 4,5 , however, only very limited information
is known about the occurrence of conjugated forms of
trichothecenes, especially DOn in processed cereal-based
foodstuff. The current study concern with analysis of DOn-
3-Glc in bread and beer is aiming at fillig the gap in this area.
The aim of study was to determine: (i) the influence of baking
technological times on levels of mycotoxins in bread and (ii)
the average contamination of commercially available beers.
Experimental
C h e m i c a l s a n d R e a g e n t s
Pure crystalline standards of analysed mycotoxins
deoxynivalenol (DOn), DOn-3-Glucoside (DOn-3-Glc),
3-acetylDOn and 15-acetylDOn (ADOns), nivalenol (nIV),
fusarenon-X (Fus-X) HT-2 toxin (HT-2), T-2 toxin (T-2) and
zearalenone (ZOn) were purchased from Biopure (Austria).
Organic solvents in HPLC grade used for LC-MS/MS analysis
were products of Sigma-Aldrich (Germany). Cellite
for beer samples purification was obtained from Sigma-Aldrich
as well. Ultra-pure water was produced by Milli-Q systém.
Working composite standards stock solution prepared
s694
in acetonitrile at concentration 1,000 ng ml –1 was stored at
4 °C. Matrix-matched standards used for analysis were prepared
by procedure described below (see sample preparation)
at concentration range 1–1,000 ng ml –1 .
S a m p l e s
Bread samples (n = 9) were prepared in laboratory scale
from whole-wheat flour, which was ground from naturaraly
infected wheat grains. The loaves of bread were prepared in
3 variants differing in times of proofing, fermentation and
baking (variant 1: 40 min. proofing and fermentation, variant
2: 45 min. proof. + ferment., variant 3: 50 min. proof. + ferment).
For the dough-making process flour (300 g), yeasts
(5.4 g), salt (4.5 g), saccharose (6 g), vegetable oil (4.5 g) and
distilled water (150–165 ml) were used. Immediately after
farinographic kneading the dough was placed into the laboratory
thermostat for 40 or 50 min. After the proofing the dough
was divided into three pieces that were allowed to ferment
for 40 or 50 min. The bread loaves were baked in an electric
laboratory oven at 240 °C for 15 or 20 min.
23 light and 7 dark beer samples, which were collected
in Czech retail market in the year 2008. All of beers analysed
within this monitoring study were derived only from barley
malts and contained 4–6 volume % of alcohol.
S a m p l e P r e p a r a t i o n
Whole-wheat flour and bread samples after the drying
were processed as follows: 12.5 g of homogenised samples
were extracted with 50 ml of acetonitril-water mixture
(84 : 16, v/v) for 30 min using an automatic laboratory shaker
(IKA Laboratortechnik, Germany). Crude extract was then
filtered (Filtrak no.390, VEB Freiberger, Germany) and 4 ml
aliquot were evaporated to dryness, the residue was transfered
into 1 ml of mobile phase in HPLC grade, consisted
of water-methanol (1 : 1, v/v) and passed through a 0.2 μm
microfilter (Alltech, USA).
The extraction step of beer samples was carried out by
analogous procedure. To 16 ml of degassed beer were added
3.2 g of Cellite and 84 ml of acetonitrile. This heterogenous
mixture was shaken for 30 min and then filtered. 5 ml
of extract were evaporated to dryness and residue again redissolved
in mixture of methanol-water (1 : 1, v/v).
L C - M S / M S M y c o t o x i n s A n a l y s i s
Separation and quantification of target analytes were
carried out by means of procedure described in detail in our
previous study 2 . Briefly, HPLC separation (HP 1100 LC system,
Agilent Technologies, USA) with reversed phase hyphenated
to tandem mass spectrometer (Finnigan LCQ Deca,
USA) were used for chromatographic separation of analytes.
Mobile phase consisted of methanol and water acidified with
ammonium acetate. Further MS/MS identification and quantification
of analytes was carried out by ion trap analyzer with
APCI ion source in both positive and negative mode.