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.     

Fate of Ochratoxin A and Citrinin During Malting and Brewing Experiments

The fate of ochratoxin A and citrinin during malting and brewing processes was studied by the use of naturally contaminated lots of barley, as well as by the addition of crystalline toxins to the mash. Complete degradation was observed for ochratoxin A from moderately contaminated barley lots and for citrinin added to mash. The use of highly contaminated barley resulted in transmission of ochratoxin A into the beer, but only 2 to 7% of the initial content was detected, corresponding to levels of 6 to 20 mug of ochratoxin A per liter of beer. Barley lots with this high ochratoxin contamination (1,000 to 5,000 mug/kg) will be easily detected and, therefore, because of pronounced deterioration, should be rejected during inspection upon admittance to the breweries.     

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine.

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznań, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.     

Mycotoxic porcine nephropathy and spontaneous occurrence of ochratoxin A residues in kidneys and blood of Polish swine

Kidneys showing renal changes characteristic for mycotoxic porcine nephropathy were collected during the period 1 April 1982 to 31 March 1983 from 225,000 swine processed in a large slaughterhouse in the district of Poznań, Poland. Of 113 kidneys suspected of mycotoxic porcine nephropathy, 27 exhibited ochratoxin A levels from traces to 23 ng/g. In 17 kidneys the level of the toxin was lower than 2 ng/g. Increased frequency of ochratoxin A presence and its level in kidneys were observed during the spring. Of 195 porcine blood samples collected at random, 36 exhibited toxin levels from 3 to 270 ng/ml.     

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.     

A survey of porcine kidneys and chicken liver for ochratoxin A in Spain

Contamination studies by ochratoxin A on pork kidney and chicken liver has been carried out in Catalonia (Spain). 73% of the pork kidney samples analyzed did not contain an amount of ochratoxin A over our detection limit (0.5 ng/g) whereas only 7% had contamination higher than 1 ng/g. None of the chicken samples analyzed were contaminated by this toxin above the detection limit. All contamination levels found are below the maximum levels accepted by several countries for this kind of material. A confirmative test is necessary before discarding false positive samples.     

Production of Ochratoxins in Different Cereal Products by Aspergillus ochraceus

The effects of temperature and length of incubation on ochratoxin A production in various substrates were studied. The optimal temperature for toxin production by Aspergillus ochraceus NRRL-3174 was found to be around 28 C. Very low levels of ochratoxin A are produced in corn, rice, and wheat bran at 4 C. The optimal time for ochratoxin A production depends on the substrate, ranging from 7 to 14 days at 28 C. Ochratoxin B and dihydroisocoumaric acid, i.e., one of the hydrolysis products of ochratoxin A, were produced in rice but at levels considerably lower than ochratoxin A. No ochratoxin C was produced in rice at 28 C. When added to rice cereal or oatmeal, the toxin was found to be very stable over prolonged storage and even to autoclaving for 3 hr.     

Nephrotoxicity of Dietary Ochratoxin A in Broiler Chikens

Graded doses of pure ochratoxin A (0,0.5,1.0,2.0,4.0, and 8.0 mug of toxin per g of feed) were incorporated into a commercial diet which was fed to chicks from 1 day to 3 weeks of age, at which time the experiments were terminated. Growth was inhibited at 2.0 4,0, and 8.0 mug/g, whereas the kidneys were enlarged at doses of 1.0 mug/g and above. Renal function as measured by clearance of phenol red was decreased 15 and 31% by doses of 4.0 and 8.0 mug/g, respectively. Uric acid was increased 38 and 48% over the control values by doses of 4.0 and 8.0 mug/g, respectively. The plasma electrolytes Na, Cl,Ca, and K were measured; however, only K was significantly ( P smaller than 0.05) altered, showing a decrease at doses of 4.0 and 8.0 mug/g. The percentage dry weight of the kidneys decreased significantly at dose levels of 4.0 and 8.0 mug/g, indicative of edema. Histological examination of kidney sections gave the impression of edema and some tubular necrosis. Pathological changes were observed at all dose levels. These data demonstrate that ochratoxin A is a severe nephrotoxin in young broiler chickens.     

Occurrence of trichothecenes, zearalenone and ochratoxin A in cereals and mixed feed from central Lithuania

A total of 92 samples — 23 winter wheat, 12 summer barley, 5 oats and 52 mixed feed — were collected from a state factory in Kaunas, Lithuania and were analysed for the presence of trichothecenes, zearalenone (ZEN) and ochratoxin A (OA) using gas chromatography with electron capture detection and immunoaffinity column/high performance liquid chromatography with fluorescence and UV detections. Deoxynivalenol (DON), nivalenol (NIV), T-2 toxin and HT-2 toxin were detected at concentrations above 10 μg/kg in 68%, 48%, 38% and 8% of cereal samples, respectively, and in 98%, 88%, 12% and 8% of samples of mixed feed for swine and poultry. More than 10 μg/kg of zearalenone and ochratoxin A were found in 58% and 92% of the mixed feed samples, respectively. The highest concentrations of all analysed trichothecenes in Lithuanian mixed feed and cereal grains, with an exception of T-2 toxin in one oat lot and one sample of mixed feed and OA in two mixed feed samples, were lower than those reported as Lithuanian advisory or tolerance limits.     

Ochratoxin A: Contamination of grain

Ochratoxin A was quantitatively monitored in grain extracts by indirect solid-phase enzyme immunoassay with the use of an immobilized conjugate of the toxin with gelatin and polyclonal rabbit antibodies raised against the ochratoxin A-BSA conjugate. This monitoring found that 1.7 to 18.5% of the samples were contaminated with the toxin at a concentration of 25.9–291.7 μg/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 μg/kg.     

Ochratoxin A in Brewer’s Yeast Used as Nutrient Supplement

Brewer’s yeast comprises different strains ofSaccharomyces cerevisiae used for beermaking. It is additionally used as a nutrient supplement to increase the intake of B vitamins and is recommended primarily for children in growth, women during pregnancy and lactation and persons during convalescence. A total of 51 samples of brewer’s yeast from the German market were analysed for the occurrence of ochratoxin A (OTA) by means of immunoaffinity clean up and HPLC with fluorescence detection. Thirty-two samples (63%) were found to be naturally contaminated with OTA in the range from the detection limit (0.03) to 1.53 ng/g. Mean values of the positive samples varied between 0.10 ng/g (powder) and 1.2 ng/g (dragees). In a worst case scenario, the consumption of brewer’s yeast could enhance the calculated daily intake for the German population by 10 to 14 ng OTA/day and person and increase the intake particularly for children from 1.3 up to about 1.9 ng/kg body weight.Thus, the results document that food supplements consisting of natural brewer’s yeast from the brewing process are a yet unknown source for the intake of ochratoxin A and a potential exposure risk. The screening of brewer’s yeast food supplements for OTA is therefore recommended in the context of food safety and quality control.     

Ochratoxin A as the cause of spontaneous nephropathy in fattening pigs.

At a number of slaughters nephropathy and high ochratoxin A contents in kidneys have been observed in fattening pigs from two Swedish farms. In one herd the source of contamination was barley grown on the home farm and stored under such conditions that the growth of fungal species (Penicillium verrucosum var. verrucosum) producing ochratoxin A occurred, with the subsequent formation of the toxin. In this case high ochratoxin A levels in fattening pigs were found during a period of about 18 months. In the second herd, where compounded feed was used, it was impossible to locate the source of contamination. It was presumed that a consignment of feed was damaged by rain during storage at the farm. Ochratoxin A was found in fattening pigs from this herd for a period of about 2 months. Ochratoxin A appeared in the kidneys of all investigated pigs. In some animals the livers, whole blood, and plasma were analyzed, too. The livers contained somewhat lower amounts of ochratoxin A than the kidneys, whereas the content in whole blood and plasma, respectively, was 5 and 13 times greater. Kidneys spontaneously contaminated with ochratoxin A, when stored for 10 months at -70 degrees C, showed no systematic decrease in toxin content.     

Solid phase extraction on sax columns as an alternative for ochratoxin A analysis in maize

A sensitive and reliable method is described for the determination of ochratoxin A (OTA) in maize. An extraction and clean-up procedure was used, with chloroform-phosphoric acid as the extractant, and liquid-liquid partition and anion-exchange chromatography (SAX columns) for the clean-up. Quantification of toxin is achieved by high performance liquid chromatography (HPLC). Recoveries were between 81-94 % at 3-90 ng/g levels. The detection limit was 0.02 ng.     

Entwicklung eines hochempfindlichen Enzymimmuntests zum Nachweis von Ochratoxin A

Antibodies against ochratoxin A were produced in rabbits after immunization with an ochratoxin A-keyhole limpet hemocyanine conjugate. The immunogen was found to be very efficient, and high antibody titers were detected in the sera of all immunized rabbits. In a competitive enzyme immunoassay using ochratoxin A-horseradish peroxidase as the labelled antigen, low levels of ochratoxin A in buffer solution could be detected. The mean standard curve detection limit and 50% inhibition level of the optimized assay were at 15 pg/ml and 50 pg/ml, respectively. Relative cross-reactivity of ochratoxin B was found to be 2%. With these characteristics, this novel enzyme immunoassay should be useful for the routine detection of ochratoxin A in food and in biological samples at levels well below 1 ng/g.     

Bioproduction of [14C]ochratoxin A in submerged culture.

A number of Aspergillus and Penicillium species were tested for production of ochratoxin A (OA) in several media. After 8 days of static incubations of submerged cultures at 28 degrees C, toxin yields of 25 and 30 micrograms/ml were obtained with Aspergillus alliaceus NRRL 4181 in Ferreirás and 2% yeast extract-4% sucrose media, respectively. However, the largest production observed in the preliminary screening was 54 micrograms/ml; this highest level was produced by A. sulphureus NRRL 4077 in a modified Czapek solution. The medium contained the basal salts and sucrose of Czapek plus urea (3%) and corn steep liquor (0.5% solids). A time study of toxin production demonstrated maximum yield of 350 micrograms/ml by the A. sulphureus isolate in the modified Czapek medium after 11 days of static incubation at 28 degrees C. The optimal production conditions were employed in additional tests designed to measure the efficiency of 14C incorporation from sodium [1-14C]-acetate into OA. Samples (20 microCi) of sodium acetate were added to separate culture flasks at 24-h intervals during the initial 9 days of the fermentation. Addition of [14C]acetate on day 4 of incubation provided the maximum yield of labeled OA. The highest specific activity of labeled toxin obtained was 0.07 microCi/mg of OA and the maximum incorporation rate of labeled acetate was 5.3%.     

Occurrence of fungi and mycotoxins in corn silage, Jalisco State, Mexico

The aim of this study was to evaluate the chemical composition, mold count and mycotoxin contamination of corn silage collected during a six month-period. The results indicated that the chemical composition and the physicochemical parameters evaluated did not show significant variation during the sampling time. Fungal count on RBC ranged from 1.7 x 10(3) to 9 x 10e8 CFU/g. Mucor, Penicillium and Aspergillus spp. were the most frequent fungal species in the corn silage. Fusarium count ranged from 1.6 x 10(3) to 1.6 x 10e8 CFU/g in Nash Snyder culture media. Aflatoxin B, fumonisins, ochratoxin A, ochratoxin B, deoxynivalenol, and zearalenone were detected throughout the period of corn silage maintenance (100% positive samples). However, only deoxynivalenol levels were higher than the maximum limit recommended by the FDA.     

Vorkommen von ochratoxin A und B in gewürzen

A total of 681 samples of spices, which comprised more than 50 different spice commodities were analysed for the natural occurrence of the mycotoxins ochratoxin A (OTA) and ochratoxin B (OTB). The analytical method involved chloroform extraction, clean-up by immunoaffinity column and HPLC determination of both mycotoxins. OTA and OTB were detected in 143 (21%) and 68 (10%) of the samples, respectively. The highest frequency of occurrence of both mycotoxins detected were in chili (100% for OTA and 55% for OTB), paprika (41% and 15%, respectively) and pepper (23% and 44%, respectively). The toxin concentrations ranged between the detection limit (0.01 ng/g) and 41.8 ng OTA (2.7 ng OTB)/g of chili, 18.9 ng OTA (1.4 ng OTB)/g of paprika and 3.8 ng OTA (4.6 ng OTB)/g of pepper. One sample of a extract of vanilla was found to be positive for OTB at 15 ng/g. However, median values of most samples showed to be below the detection limit. Comparison of the geographical origin of the samples showed that the predominant number of contaminated spices was from Southeast-Asia and India. Highly contaminated paprika samples were found to come from Israel.     

Secondary fungal metabolites (mycotoxins) in lichens of different taxonomic groups

Secondary fungal metabolites (mycotoxins) in 22 lichen species of the families Parmeliaceae, Nephromataceae, Umbilicariaceae, Ramalinaceae, Cladoniaceae, Peltigeraceae, and Teloschistaceae were determined by enzyme-linked immunosorbent assay. The following mycotoxins were found in these lichens in a broad concentration range with a frequency of 70–100%: sterigmatocystin (7–2090 ng/g), alternariol (20–6460 ng/g), and emodin (45–94500 ng/g). Mycophenolic acid frequently occurred in 19 lichen species; citrinin, in 17 species; diacetoxyscirpenol, in 11 species; cyclopiazonic acid, in 10 species; and zearalenone, in 9 species. PR toxin was regularly detected in three lichen species; deoxynivalenol, fumonisins, and ochratoxin A, in two species; and T-2 toxin and ergot alkaloids, in one species. Aflatoxin B₁ was detected in only six species with a frequency of 2–42%, whereas roridin A was present in 10% of Hypogymnia physodes samples.     

Biochemical changes in the rat during experimentally induced acute ochratoxicosis

Biochemical changes in rat urine and tissues treated with five consecutive daily doses of ochratoxin A (10 mg/kg body weight) were studied. Urine volume and urinary proteins were moderately raised during the first few days of ochratoxin treatment, and were then highly elevated towards the end of the investigation. Urinary muramidase excretion was significantly raised (p less than 0.01) 24 h after the first insult with the toxin. The urinary output of alkaline and acid phosphatases, lactate dehydrogenase (LDH) and glutamate dehydrogenase (GDH) were all elevated but very much later, during the course of injections with ochratoxin A. Kidney alkaline and acid phosphatases, LDH and GDH were correspondingly reduced 7 days from the beginning of ochratoxin A administration. Liver LDH activity was reduced while serum LDH was raised. Liver glycogen level was significantly (p less than 0.0001) increased. Experimental evidence was presented to show that the initial point of interaction of ochratoxin A with the rat renal system may be at the first portion of the proximal convoluted tubular cell region.     

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.