Natural occurrence of ochratoxin A and citrinin in food stuffs in Egypt

Natural occurrence of ochratoxin A (OA) and citrinin in cereals (274 samples) and animal tissues (250 samples) have been investigated during a period of more than 2 years. OA was found in cereals and animal tissues while citrinin was found in cereals only. The highest level of OA (up to 80.0 μg/kg) was found in yellow corn, 52.8% of contaminated samples while respectively 55.9% and 39.4% of barley and rice samples were contaminated with citrinin, with the highest level up to 100.0 and 27.92 μg/kg for barley and rice respectively. The frequent contamination of animal kidney with OA (28% positive out of 150 tested) average concentration 12.33 μg/kg. 2% of liver and 4% of muscles tissue were observed.     

Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing penicillia.

In a batch of barley associated with field cases of mycotoxic porcine nephropathy and containing ochratoxin A and citrinin, the mycoflora were isolated by parallel incubation at 10 and 25 degrees C. Subsequently, the isolated cultures were checked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin, and citrinin). The nephrotoxin producers, all isolated by incubation at 10 degrees C, were comprised of one culture of Penicillium viridicatum, five cultures of Penicillium cyclopium, and one culture of Penicillium crustosum, all producing xanthomegnin and viomellein. One culture of P. cyclopium produced citrinin. Viomellein was detected in the barley at a concentration of approximately 1 mg/kg. The method of analysis for xanthomegnin and viomellein included extraction with chloroform, partitioning in hexane-acetone, and thin-layer chromatographic separation and identification. The identity of the xanthomegnin and viomellein produced by the isolated fungi and of viomellein detected in the barley was supported by infrared spectroscopy. This is the first report of viomellein as a natural contaminant of foodstuffs.     

Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing penicillia

In a batch of barley associated with field cases of mycotoxic porcine nephropathy and containing ochratoxin A and citrinin, the mycoflora were isolated by parallel incubation at 10 and 25 degrees C. Subsequently, the isolated cultures were checked for production of nephrotoxins (xanthomegnin, viomellein, ochratoxin, and citrinin). The nephrotoxin producers, all isolated by incubation at 10 degrees C, were comprised of one culture of Penicillium viridicatum, five cultures of Penicillium cyclopium, and one culture of Penicillium crustosum, all producing xanthomegnin and viomellein. One culture of P. cyclopium produced citrinin. Viomellein was detected in the barley at a concentration of approximately 1 mg/kg. The method of analysis for xanthomegnin and viomellein included extraction with chloroform, partitioning in hexane-acetone, and thin-layer chromatographic separation and identification. The identity of the xanthomegnin and viomellein produced by the isolated fungi and of viomellein detected in the barley was supported by infrared spectroscopy. This is the first report of viomellein as a natural contaminant of foodstuffs.     

Fate of ochratoxin A in brewing.

The fate of ochratoxin A in brewing was investigated by adding (3H)ochratoxin A to the raw materials at 1- and 10-mug/g levels during mashing in a conventional microbrewing process. The results indicated that large portions (28 to 39%) of the added toxin were recovered in spent grains, with less recovery in the yeast (8 to 20%) and beer (14 to 18%). About 38 and 12% of the added toxin at levels of 1 and 10 mug/g, respectively, were degraded during brewing.     

Detection of citrinin in ochratoxin A-containing products by a new HPLC method

A new method for citrinin was developed and validated, which is based on solid phase extraction with polyamide columns and HPLC with fluorescence detection. Sufficient skill with the method given, precise results, i.e. variation coefficients <10%, will be achieved. The mean recovery rates were in the range 74 – 90%. The detection limits of the method determined according to DIN 32645, at good precision, were 1 μg/kg for wheat, rye, barley, maize, and oats. The analysis of several samples containing ochratoxin A (OTA) showed that citrinin is present in brans, wheatings and shorts containing a higher ratio of the outer layers of the grain kernel; both OTA and citrinin were found in in cocoa shells and raisins. Citrinin was detected in 14 OTA-containing samples (1–8 μg/kg). Furthermore, it was demonstrated that citrinin also can be determined in red mold rice according to the new method. Presented at the 25^th Mykotoxin Workshop in Giessen, Germany, May 19–21, 2003     

Toxic effects of ochratoxin A and citrinin, alone and in combination, on chicken embryos.

The embryotoxic potential of ochratoxin A and citrinin was studied after administering, either subgerminally or intraamniotically, single mounting doses of the mycotoxins to chicken embryos on days 2, 3, and 4. The beginning of the embryotoxicity dose range was found to be between 0.01 to 0.05 microgram for ochratoxin A and 1 to 10 micrograms for citrinin. The maximum response to both mycotoxins occurred after administration on day 3. In addition to significant growth retardation of fetuses, exencephaly, microphthalmia, cleft beak, reduction deformities of the limbs, and abdominal wall and ventricular septal defects were encountered on day 8 of incubation. When 4 micrograms of citrinin was constantly added to ochratoxin A administered in the dose range of 0.03 to 0.5 microgram, a strictly additive effect was seen. It may be supposed that citrinin produced together with ochratoxin A in some strains of Penicillium viridicatum Westling does not potentiate the clear-cut embryotoxic action of the latter mycotoxin.     

Toxic effects of ochratoxin A and citrinin, alone and in combination, on chicken embryos

The embryotoxic potential of ochratoxin A and citrinin was studied after administering, either subgerminally or intraamniotically, single mounting doses of the mycotoxins to chicken embryos on days 2, 3, and 4. The beginning of the embryotoxicity dose range was found to be between 0.01 to 0.05 microgram for ochratoxin A and 1 to 10 micrograms for citrinin. The maximum response to both mycotoxins occurred after administration on day 3. In addition to significant growth retardation of fetuses, exencephaly, microphthalmia, cleft beak, reduction deformities of the limbs, and abdominal wall and ventricular septal defects were encountered on day 8 of incubation. When 4 micrograms of citrinin was constantly added to ochratoxin A administered in the dose range of 0.03 to 0.5 microgram, a strictly additive effect was seen. It may be supposed that citrinin produced together with ochratoxin A in some strains of Penicillium viridicatum Westling does not potentiate the clear-cut embryotoxic action of the latter mycotoxin.     

The fate of mycotoxins during the distillation process of barley shochu, a distilled alcoholic beverage

Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.     

Production of ochratoxin A in barley by Aspergillus ochraceus and Penicillium viridicatum: effect of fungal growth, time, temperature, and inoculum size.

Moistened barley was inoculated with 1.4 x 10(3) and 1.4 x 10(5) spores, respectively, from ochratoxin A-producing strains of Aspergillus ochraceus and Penicillium varidicatum. To estimate fungal tissue in the barley, the amount of glucosamine was followed for 28 days at 10 and 25 degrees C. Ochratoxin A was also followed during the same period and under the same conditions. The data show that ochratoxin A could be detected 4 to 6 days after inoculation at 25 degrees C, and the maximal accumulation of ochratoxin A was observed 28 days after inoculation. After 28 days at 25 degrees C, the quantities of ochratoxin A were between 7 and 46 micrograms/g of grain. At 10 degrees C only P. viridicatum produced ochratoxin A. The results indicated that production of ochratoxin A is not associated with rapid increase of glucosamine in the barley.     

The Fate of Mycotoxins during the Distillation Process of Barley Shochu,a Distilled Alcoholic Beverage

Mycotoxins are frequent contaminants of grains and critical risk substances for brewers. Fermented barley mash contaminated artificially with 13 representative mycotoxins was distilled with small-scale apparatuses to elucidate the possibility of mycotoxin transfer from mash to distillates. None of these were detected in the distillates. The distillation process can effectively reduce the contamination risk posed by mycotoxins in distilled alcoholic beverages.     

Citrinin,ochratoxin A and iron. Possible implications for their biological function and induction of nephropathy

Experiments with Neisseria meningitidis have shown that Fe³⁺ to some extent can reverse the toxicity of ochratoxin A and citrinin, as measured by inhibition zones around impregnated paper discs. Similar phenomena were observed with the less toxic ochratoxin B. Zearalenone also inhibited growth, but its effect was not counteracted by iron. The mycotoxins aflatoxin B₁ and deoxynivalenol did not inhibit bacterial growth at all. Desferal (deferoxamine) also inhibited growth of meningococci, but iron totally abolished this inhibition. The results indicate that ochratoxin A and citrinin interfere with iron metabolism in this organism but that other additional toxic mechanisms are involved as well since a marked growth inhibition by both toxins was also observed in the presence of iron. One function of ochratoxin A and citrinin in nature could consequently be to affect the iron uptake of other competing microorgansms.Since both toxins interfere with iron and both cause nephropathy, a possible connection between these properties and lipid peroxidation is also briefly discussed.Abbreviations DON deoxynivalenol - OA ochratoxin A - OB ochratoxin B     

Brine Shrimp (Artemia salina L.) Larvae as a Screening System for Fungal Toxins

Concentrations resulting in 50% mortality, determined with brine shrimp (Artemia salina L.) larvae exposed to known mycotoxins for 16 hr, were (mug/ml): aflatoxin G(1), 1.3; diacetoxyscirpenol, 0.47; gliotoxin, 3.5; ochratoxin A, 10.1; and sterigmatocystin, 0.54. 4-Acetamido-4-hydroxy-2-butenoic acid gamma-lactone gave no mortality at 10 mug/ml. Used as a screening system involving discs saturated with solutions of known mycotoxins, the larvae were relatively sensitive to aflatoxin B(1), diacetoxyscirpenol, gliotoxin, kojic acid, ochratoxin A, rubratoxin B, sterigmatocystin, stemphone, and T-2 toxin. Quantities of 0.2 to 2 mug/disc caused detectable mortality. The larvae were only moderately sensitive to citrinin, patulin, penicillic acid, and zearalenone which were detectable at 10 to 20 mug/disc. They were relatively insensitive to griseofulvin, luteoskyrin, oxalic acid, and beta-nitropropionic acid. The disc screening method indicated that 27 out of 70 fungal isolates from foods and feeds grown in liquid or solid media produced chloroform-extractable toxic material. Examination of toxic extracts by thin-layer chromatography for 17 known mycotoxins showed that the toxicity of eight isolates could be attributed to aflatoxin B(1) and B(2), kojic acid, zearalenone, T-2 toxin, or ochratoxin A. Nine out of 32 of these fungal isolates grown in four liquid media yielded toxic culture filtrates from at least one medium. Chemical tests for kojic, oxalic, and beta-nitropropionic acids showed the presence of one or two of these compounds in filtrates of seven of these nine isolates.     

Degradation of ochratoxin a and b by the white rot fungus<Emphasis Type="Italic">pleurotus ostreatus</Emphasis>

Degradation of ochratoxin A (OTA) and B (OTB) by three selected fungi during solid state fermentation of barley contaminated with ochratoxins was compared. In presence of the soil fungusRhizopus japonicus and the white rot fungusPanerochaete chrysosporium more than 60 % of the mycotoxins remained stable, while in the white rot fungusPleurotus ostreatus only 23 % of the initial OTA and 3 % of OTB were detected after a four weeks incubation period. Kinetic studies on mycotoxin degradation byPI ostreatus demonstrated formation of ochratoxin α and presumably ochratoxin β as intermediate products, what indicates that hydrolysis is the first step in OTA and OTB degradation followed by further degradation of the intermediates.     

Lack of mutagenicity of ochratoxin A and B, citrinin, patulin and cnestine in Salmonella typhimurium TA102.

The Aspergillus mycotoxins ochratoxin A and B, citrinin and patulin as well as combinations of ochratoxin A and citrinin did not induce reverse mutations in Salmonella typhimurium strain TA102. Therefore there is no indication for the induction of oxidative damage or crosslinks. The same is true for cnestine, a compound extracted from the plant Cnestis glabra.     

Nachweis und Vorkommen von Mykophenolsäure und Citrinin in Rotwein

Due to infections with moulds already in the vineyards, the formation of mycotoxins is possible under certain circumstances during the process of red wine making. At this, metabolites ofPenicillium spp. are of major importance, as this species are to be found frequently on grapes. Beside the nephrotoxic citrinin, which is often co-occurring with ochratoxin A, the occurrence of mycophenolic acid (MPA), a substance of immunosuppressive action, was investigated since it is formed by a great number of Penicillium-species. The detection of these compounds was carried out by means of ELISA and LC-MS. As testing material 44 red wine samples of different provenience and vintages were used. Mycophenolic acid could be detected in 91 % of the samples. The maximum content amounted to 130 ng/ml, yet most of the samples resulted in much lower concentrations of between 3 and 20 ng/ml. The extent of contamination seems to depend rather on the origin of the wine than on the vintage. In particular samples from Southern Europe were most contaminated. This could be due to different practises in wine-making. Citrinin was not detectable in any sample (< 0,2 ng/ml). Regarding the detected concentrations of MPA and citrinin, there is probably no concern for consumers’ health. However, the degree of contamination of wine with MPA may well serve as an indicator for hygiene in production.     

Mycotoxins and toxigenic fungi in sago starch from Papua New Guinea

AIMS: To assay sago starch from Papua New Guinea (PNG) for important mycotoxins and to test fungal isolates from sago for mycotoxin production in culture. METHODS AND RESULTS: Sago starch collected from Western and East Sepik Provinces was assayed for aflatoxins, ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin and zearalenone and all 51 samples were negative. Frequently isolated species of Penicillium (13), Aspergillus (five) and Fusarium (one) were cultured on wheat grain, and tested for the production of ochratoxin A, cyclopiazonic acid, sterigmatocystin, citrinin, patulin and penicillic acid. All 12 isolates of P. citrinin and one of two A. flavipes isolates produced citrinin. A single isolate of A. versicolor produced sterigmatocystin. No other mycotoxins were detected in these cultures. CONCLUSIONS: No evidence was found of systemic mycotoxin contamination of sago starch. However, the isolation of several mycotoxigenic fungi shows the potential for citrinin and other mycotoxins to be produced in sago stored under special conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Sago starch is the staple carbohydrate in lowland PNG and the absence of mycotoxins in freshly prepared sago starch is a positive finding. However, the frequent isolation of citrinin-producing fungi indicates a potential health risk for sago consumers, and food safety is dependant on promoting good storage practices.     

Stability of citrinin and deoxynivalenol during germination process of barley

The stability of citrinin and deoxynivalenol during germination process of barley spiked with these mycotoxins at a level of 2/ig/g was investigated. Germinated barley was analyzed after 1, 3, and 5 days to follow the stability of citrinin and deoxynivalenol during the germination process. Two thin layer chromatographic methods were used for determinations of citrinin and deoxynivalenol. An average of 93.6% of citrinin and 77.1% of deoxynivalenol were destroyed within 5 days during the germination process of barley.     

Mycotoxin formation in moist 2-row and 6-row barley during granary storage

Eleven-kilogram parcels of 2-row and 6-row barley initially at 18% moisture content were implanted in dry bulk oats in a farm granary in Manitoba for 60 weeks between August 1983 and October 1984. Temperature, moisture content, O₂ and CO₂ levels, fat acidity values, seed germination, microfloral incidence and abundance and the presence of major mycotoxins (aflatoxins, sterigmatocystin, ochratoxin A, citrinin, penicillinic acid, patulin) were monitored. Ochratoxin A reached maximum levels of 0.97 ppm by week 24 in the 6-row barley, and 0.05 ppm by week 28 in the 2-row; no other mycotoxins were detected. The effect of cultivar type was significant (P<0.01) with greater effects in the 6-row barley for the following parameters: fat acidity value, germination, incidence of Alternaria alternata, Penicillium spp. and Helminthosporium sativum, total fungal propagule count and ochratoxin A levels. The effect of time was significant (P<0.05) for all variables except oxygen, carbon dioxide, Aspergillus versicolor, and total fungal propagule count. The interaction between cultivar and time was significant (P<0.01) for Alternaria and Helminthosporium only.Contribution No. 1233, Agriculture Canada Research Station, Winnipeg, Manitoba R3T 2M9, Canada.     

Stability of Aflatoxin B1 and Ochratoxin A in Brewing

The stability of aflatoxin B₁ and ochratoxin A in brewing was investigated by adding the purified toxins to the raw materials at 1 and 10 μg/g levels during mashing in a conventional micro-brewing process. The results indicate that both toxins are stable to heat and are insensitive to cooker mash treatment. Both mycotoxins were partially removed in the mashing and brewing processes. About 14 to 18% and 27 to 28% of the added toxins were found in the final beers brewed from starting materials containing 1 and 10 μg, respectively, of either toxin per g. The possible route of transmission of mycotoxins into beer is discussed.     

Analysis of mycotoxins in cereals and animal feed using a multi-toxin gel permeation chromatography clean-up method

Gel permeation chromatography has been used to clean up extracts from cereals and animal feeds containing a range of mycotoxins. A mixture of dichloromethane: IM hydrochloric acid, 10:1 by volume is used as the extraction solvent and clean-up is carried out on a Bio — Beads S-X3 column using dichloromethane: ethyl acetate containing a small amount of formic acid as the elution solvent. Chromatographic separation and detection was by HPLC with fluorescence or ultraviolet detection although the choice of detection method is left to the user. The method has been tested for 14 mycotoxins and results are presented for cereals fortified with mycotoxins and for samples naturally contaminated with aflatoxins, citrinin, zearalenone, and ochratoxin A.