Occurrence ofAlternaria andFusarium mycotoxins in winter wheat from domestic crop in year 2003.

The aim of this study was a monitoring of the occurrence ofAlternaria andFusarium mycotoxins in winter wheat from domestic crop in the year 2003. Altenuene was determined in 56 (100%) samples of winter wheat, range 14.5-41 µg/kg, mean 25 µg/kg. Alternariol was determined in 16 (28.6%) samples of winter wheat, range 6.3-22.1 µg/kg, mean 5.7 µ/kg. DON was determined in 42 (100%) samples of winter wheat, range 250-3500 µg/kg, mean 330 µg/kg. T2-toxin was determined in 42 (100%) samples of winter wheat, range 25-337 µg/kg, mean 99 µg/kg. ZEA was not determined in samples of winter wheat.

Monitoring of mycotoxin biomarkers in the Czech Republic.

AFM1 was determined in 72 (72%) samples of human urine, range 19-6064 pg/g creatinine, mean 367 pg/g creatinine, median 158 pg/g creatinine and 90% percentile 755 pg/g creatinine in 1997. AFM1 was determined in 46 (43.8%) samples of human urine, range 21-19219 pg/g creatinine, mean 414 pg/g creatinine, median 96 pg/g creatinine and 90% percentile 415 pg/g creatinine in 1998. OTA was determined in 2077 (94.2%) samples of human serum, range 0.1-13.7 µg/L, mean 0.28 µg/L, median 0.2 µg/L and 90% percentile 0.5 µg/L in 1994-2002. OTA was determined in 12 (40%) samples of human kidneys, range 0.1-0.2 µg/kg, mean 0.07 µg/kg, and median 0.05 µg/kg in 2001.

[Monitoring the important mycotoxin biomarkers (ochratoxin A, aflatoxin M1) in the Czech population]. / Stav sledováni významných biomarkeru mykotoxinu (ochratoxinu A, aflatoxinu M1) u populace v Ceské republice.

BACKGROUND: In among mycotoxins, secondary metabolites of toxinogenic moulds, ochratoxin A and aflatoxins occupy a prominent place. These mycotoxins have been--as etiologic agents-- associated with a wide numbers of acute and chronic human diseases including mycotoxicoses like Balkan endemic nephropathy or liver cancer. While the risk of acute toxic effects of ochratoxin A and aflatoxin B1 is usually considered to be minimal in the Czech Republic, the situation is different as far as the risk of the late toxic effects (particularly carcinogenic), which result from a single or repeated intake of low doses of these mycotoxins from foodstuffs is concerned. METHODS AND RESULTS: The presence of ochratoxin A and aflatoxins in human environment has been monitored within the program "Monitoring the health state of the population". As for ochratoxin A, 2206 samples of blood serum were investigated, 2077 (94 %) of them turned out to be positive (with levels > or = 0.1 microg x l(-1)), the average was 0.28 microg x l(-1), the median was 0.2 microg x (l(-1), and the percentile (90%) was 0.5 microg x l(-1). The ochratoxin A levels ranged from 0.1 to 13.7 microg x l(-1) of sera. The presence of ochratoxin A was also analyzed in 30 samples of human kidneys; 12 samples were positive (with levels > or = 0.1 microg l(-2), the average was 0.07 microg x kg(-1), the median was 0,05 microg x kg(-1). As for aflatoxins, in 1997-1998 the presence of aflatoxin M1 was investigated in 205 samples of human urine; 118 samples (58%) were positive (with levels >125 pg x l(-1) of urine). CONCLUSIONS: When calculated to a concentration of creatinine in urine, the average was 391 pg x g(-1), the median was 127 pg x g(-1), and the percentile (90%) was 585 pg x g(-1). The aflatoxin M1 levels ranged from 19 to 19 219 pg x g(-1) of creatinine.

A HPTLC method for the determination of the mycotoxin zearalenone in cereal products.

An HPTLC method for the quantification of zearalenone (ZEA) in cereals and cereal products (wheat flour and malt) has been developed. ZEA was extracted with 50 ml of acetonitrile-purified water (9+1) with addition of 2 g NaCl. The extracts were further purified on VICAM ZearalaTest((tm)) immunoaffinity columns, then analysed by instrumental high-performance thin-layer chromatography (HPTLC) on silica gel plates with fluorescence detection. Ethyl acetate - n-hexane (1+1) was used as the mobile phase. The chromatogram was scanned in fluorescence mode after excitation at λ=254 nm with λ=400 nm measuring filter: SENS and SPAN parameters were 195 and 20, respectively. TheR F of ZEA under these conditions was 0.43.The recovery was 95% in the range 15-65 µg/kg cereal products; the mean relative standard deviation of repeatability (RSDr) was 7.6%. The limit of quantification (LoQ) of ZEA was 10 µg/kg. Validation of the method was performed according to the principles of the ICH for pharmaceutical analysis.

The system approach to the identification of aflatoxigenic fungi in foodstuffs and feedstuffs.

Aspergillus flavus, A parasiticus, A nomius, A tamarii andA pseudotamarii are important microorganisms capable of producing aflatoxins and further mycotoxins. Aflatoxigenic Aspergillus species are morphologically similar species belonging to the Aspergillus section Flavi. The aflatoxigenic fungal strains were isolated from foods (cereals, pulses, oilseeds, dried fruit, spices), soil, air and water. Mycological analyses are based on valid standards and recommendations of the International Commission for Food Mycology (ICFM). The identification of isolated aflatoxigenic fungi in foodstuffs and feedstuffs can be proved by using classical mycological cultivation methods, diagnostic nutrient media, chemotaxonomy and molecular biological methods (PCR). The system approach to the identification of aflatoxigenic fungi combines these four methods. Thirty strains of the aflatoxigenic fungi were tested.

The mycotoxin research in foodstuffs in the Czech Republic in the 90th Years.

Mycotoxins are naturally occurring secondary metabolites produced by several toxigenic microscopic fungi on a variety of crops, especially cereal grains and further foodstuffs. Series of experimental research projects on the determination of mycotoxins (aflatoxins, cyclopiazonic acid, ochratoxin A, patulin, deoxynivalenol, fumonisin B1, T-2 toxin, zearalenone, sterigmatocystin, alternaria toxins) in several foods were realized in the National Reference Centre for Microscopic Fungi and Mycotoxins in the 90(th) years. The aim of our work was an estimation of dietary exposure to mycotoxins and risk assessment. The method of a solid phase extraction (SPE), liquid - liquid extraction and immunoaffinity chromatography (f. e. R-Biopharm, VICAM) were used to elaborate for sample analyses of mycotoxins in our projects. The mycotoxins were detected most frequently by chromatographic methods (HPTLC, HPLC, GC) and immunochemical methods (ELISA). Average dietary exposure has been calculated by multiplying of concentration data for specific foods with their consumption rates per 1 kg of b. w. per day. The estimation of the dietary exposure dose of mycotoxins for the Czech population is presented.

Occurrence of the toxigenic fungi (producers of aflatoxins and ochratoxin A) in foodstuffs in the Czech Republic 1999-2000.

The occurrence of toxigenic fungi producing aflatoxins and ochratoxin A in foodstuffs was studied in the Czech Republic. Twenty five commodities were collected at twelve collection places in the Czech Republic (300 food samples). The presence of potentially toxigenicAspergillus flavus was observed in 28% of the sampled foods (black pepper, caraway seeds, fruit tea, black tea, oat flakes, fine flour, rolled oat flakes and semolina) in the year 1999, and in 25% of the sampled foods (black pepper, black tea, fine flour) in the year 2000.A tamarii (aflatoxins producer) was found in 3 black pepper samples (25%) in both years. Aflatoxins were detected in black pepper and caraway seed samples in the year 1999 and in sweet red pepper in the year 2000.A parasiticus andA nomius were not isolated. Aspergillus section Nigri (potential producer of ochratoxin A) was detected in some foodstuffs. Ochratoxin A was detected in raisins.Penicillium verrucosum andA ochraceus were not isolated from foodstuffs.

[Micromycetes, mycotoxins and human health]. / Mikromycety, mykotoxiny a zdraví cloveka.

Mycotoxins are toxic metabolites produced by certain toxigenic microscopic fungi (moulds) in and on foods. Consequently mycotoxin-containing foods have been found all over the world: Africa, Asia, North and South America, Australia and Europe. The extent of the problem is greater in some parts of the world than in others because their climatic conditions are more favourable for mould growth and thus synthesis of mycotoxins. These toxins have been associated with various diseases-mycotoxicoses in humans throughout the world (ergotism, alimentary toxic aleukia, aflatoxicosis, balkan nephropathy, yellow rice disease, oesophageal cancer etc.). Mycotoxins can enter the food chain by one of two major routes: direct contamination resulting from the use of a food components contaminated with mycotoxins and indirect contamination resulting from the growth of toxigenic fungi of the food. Investigations of mycotoxins in foodstuffs, in human urine and human milk were incorporated into the system of Environmental Health Monitoring in the Czech Republic. The risk of acute toxic effects of mycotoxins was usually considered to be minimal in the Czech Republic. The risk of later toxic effects (particularly carcinogenic risk) after very low single or repeated mycotoxin concentrations in foodstuffs is very important.

Determination of the mycotoxin fumonisins in gluten-free diet (corn-based commodities) in the Czech Republic.

The fumonisins, mycotoxins produced by Fusarium moniliforme, are known to occur worldwide as natural contaminants of corn. They are associated with several animal diseases and are a potential threat to human health. A total of 127 samples of corn-based foods (gluten-free diet) in the Czech Republic were analysed by Ridascreen Fumonisin Fast ELISA methods in years 1995-1996. Eighty eight % of the corn-based foods were found to be positive for fumonisins (FB1, FB2, FB3) and 12% of the examined corn-based foods laid below of a determination limit which was about 9 ng fumonisins/g corn-based foods. The highest fumonisin contamination levels were recorded in extruded corn products containing up to 1,808 micrograms/kg of fumonisins. Levels ranging from < 9 to 1,243 ng/g fumonisins were detected in polenta. Lower levels of fumonisins were found in other commodities, such as corn flour (up to 487 ng/g), corn instant porridge (up to 788 ng/g), and corn pastes (511 ng/g). Intake of fumonisins from several corn-based foods (gluten-free diet) for the population with coeliac disease was estimated. The highest estimate of exposure dose of fumonisins was determined from corn-extruded bread: 3.2 micrograms/person/day (mean of measured values). Daily intake of fumonisins from polenta is expected 2.8 micrograms/person/day (mean). The lower exposure dose of fumonisins we can expect from corn instant porridge, corn postes and other corn products--corn and amaranth biscuit, corn beverage: 0.9, 1.1 and 0.3 micrograms/person/day (mean) respectively.

Determination of the mycotoxin deoxynivalenol in beer by commercial elisa tests and estimation of the exposure dose from beer for the population in the Czech Republic.

About 150 litres of beer per person is consumed yearly in the Czech Republic. It is one of the highest consumptions in international comparison. Thus, beer can be a significant source of exposure of man to some contaminants. Among natural contaminants of beer belongs also deoxynivalenol (DON). 77 samples of beer being sold in the shopping network in the Czech Republic were examined by means of commercial ELISA sets for determination of deoxynivalenol (RIDASCREEN DON) at the end of the year 1994 and at the beginning of the year 1995. 23% of the beer samples were under a detection limit of the ELISA test (6 mu g of DON/litre). The other samples contained DON in amount of 7-70 mu g/litre. The median came up to 12.6 mu g of DON/litre. By variance analysis, statistically conclusive (P = 0.01) lower concentrations of DON were found in 11-12% light beer in comparison with 10% light beer and other beer. On the basis of results, the average exposure of beer consumers in the Czech Republic was estimated at a level of 0.146 mu g of DON/kg of b.w./day. The calculation was based on a geometric mean of DON concentrations and on an average corrected consumption of individual kinds of beer. This exposure represents about 11.7% of the proposed TDI (1.25 mu g of DON/kg of b.w./day, determined from the view of the immunosuppression effect of DON). The detected exposure to DON from beer does not represent a serious health risk for a consumer in the Czech Republic. In calculations of the total exposure dose of DON for man from cereal sources is it, however, appropriate to include also beer.

Health risk assessment of the mycotoxin ochratoxin A to humans: Czech Republic--Brno--1991/92.

In the course of year 1991 and 1992 about 594 blood donors of the Brno agglomeration in the Czech Republic were examined for the ochratoxin A content (OA) in blood serum. When higher concentrations of OA were found the blood donors were examined repeatedly (differentiation of acute or chronic exposure). A mean concentration of 0.63 microgram OA/l blood serum (0.30 microgram = geom.mean) was recorded. The assessed continuous mean daily dietary intake of OA was about 0.74 ng (0.35 ng = geom. mean) OA/kg b.w./day. The assessed continuous mean contamination of food groups (cereal and meat products) was about 0.65 microgram (0.31 microgram = geom. mean) OA/kg. In persons with elevated OA concentrations in blood serum the decrease was at the latest confirmed within 2 months after the test result. An accidental acute exposure was probably involved. Tolerable daily intake of OA (TDI) was determined with regard to the nephrotoxic, teratogenic, immunosuppressive and carcinogenic effect at the level of: 16,500, 250 and 5 ng OA/kg b.w./day. As a legislative limit TDI = 5 ng OA/kg b.w./day was suggested. The group of persons studied was probably not threatened by any of the health risks given.

Study of human exposure to ochratoxin A and assessment of possible sources.

The first study carried out in the CSFR covered 644 samples of blood sera from the district of Uherské Hradiste (about 1,000 km2 and 150,000 population) during March-May 1990. The samples from the selected persons (over 18 years of age) were collected within 4 sampling weeks. Ochratoxin A (OA) was established densitometrically after the minicolumn separation with HPTLC. The detection limit was about 0.5 microgram OA/l, recovery about 95%. The maximum established value was 12 micrograms OA/l. The value 1 microgram OA/l was exceeded by 12.4% of samples. Seventy-eight per cent of samples were under the detection limit. After stratification of the experimental group (according to sex and age) higher numbers of findings above 1 microgram OA/l were found in the stratum of 30-40 years (males and females) and in the age group over 60 (females). The differences were statistically insignificant (contingency table analysis, alpha > 0.10). The high statistical significance (contingency table analysis) of difference (alpha < 0.01) showed the findings over 1 microgram OA/l, and the date of the sampling week. The graphic analysis (localisation of the results in the map of the area) did not support the hypothesis of the dependence of the results over 1 microgram OA/l on the place of residence. The results do not support the hypothesis on the sites with a higher level of OA contamination in the studied district. We assume the OA hazard sources originating from both the individual and communal food supply.

[Long-term administration of the mycotoxin, ochratoxin A in chickens and the residues in the meat and animal feed]. / Dlouhodobý príjem mykotoxinu ochratoxin A u kurat z hlediska reziduí v potravinárských a krmných surovinách.

A feeding trial was performed with chick broilers (cockerels). The feed with an addition of 850 micrograms ochratoxin A (OA) per kg was administered for six weeks. The feeding of the chicks stopped twelve hours before slaughter (in keeping with slaughter technology for chicks). Blood, liver and kidney samples were taken. At the end of trial the level of OA residues in the samples did not exceed 5 micrograms per kg. In other trials the dynamics of OA residues in the blood plasma of chicks was investigated after i.v. implantation at an amount of 2 and 20 micrograms per chick (1.5 kg lw.). An open two-compartment model was used to estimate toxicokinetic parameters. The half-time of elimination (t1/2(beta)) was about 3.3 hours. The high total clearance (CL) of 34.2 ml/min/kg lw. and apparent distribution volume (Vd(area)) of 9.8 l/kg lw. demonstrate rapid distribution to the tissues and rapid OA elimination. The results document that neither at a long-term intake of feed contaminated to the level of 850 micrograms OA per kg will the present hygienic limits of residues for foods be exceeded (5 and 20 micrograms per kg) if the principles of correct slaughter technology are observed. The blood of chicks used as feed is not an important source of OA in this case.

[Immunoenzyme detection of the mycotoxin, ochratoxin A]. / Enzymoimunologické stanovení mykotoxinu ochratoxin A.

Indirect, enzymoimmunological assays of the mycotoxin ochratoxin A were developed. In this technique a polyclonal (rabbit) antibody to ochratoxin A was used, along with the other, peroxidase-labelled (pig anti rabbit) antibody. The sensitivity of this method ranged around 75 pg of ochratoxin A per pit. The range of calibration curve was from 10 to 1000 pg per pit. The cross reactions with other ochratoxins made 1.4% (ochratoxin C). The ELISA test of ochratoxin A can be used as an expeditious screening method for a preliminary examination of the greater number of samples.

[Detection of cyclopiazonic acid and its producers in food]. / Nálezy kyseliny cyklopiazonové a jejích producentu v pozivatinách.

In the course of six months, 60 samples of foods were examined for their contents of cyclopiazonic acid. These samples were subjected to a basal mycological screening aimed at Aspergillus flavus and Penicillium sp. strains. Cyclopiazonic acid contents in samples of Hermelín cheese, peanuts, rice, peeled barley grains, Folican salami, and packaged meat did not exceed the value of 0.5 mg.kg-1. When using a modification of the method of cyclopiazonic acid isolation described by Dorner et al. (1983), 521 mg of this mycotoxin were isolated from a culture of Penicillium griseofulvum CCM 8006 strain grown in liquid medium containing 2% yeast autolysate and 2.5% sucrose. About 47% of the isolated Aspergillus flavus strains were bitoxicogenic (produced both cyclopiazonic acid and aflatoxin). Cyclopiazonic acid was produced by 23.5% of the isolated Penicillium sp. strains. No cyclopiazonic acid was produced in vitro by Penicillium nalgoviensis strains from the Czechoslovak collection on sweet wort agar containing peptone from soybean. Penicillium commune F-426 and Penicillium aurantiogriseum F-708 strains are efficient producers of this acid.