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.     

Inhibition by antimicrobial food additives of ochratoxin A production by Aspergillus sulphureus and Penicillium viridicatum

The effects of antimicrobial food additives on growth and ochratoxin A production by Aspergillus sulphureus NRRL 4077 and Penicillium viridicatum NRRL 3711 were investigated. At pH 4.5, growth and toxin production by both A. sulphureus and P. viridicatum were completely inhibited by 0.02% potassium sorbate, 0.067% methyl paraben, 0.0667% methyl paraben, and 0.2% sodium propionate. At pH 5.5, 0.134% potassium sorbate and 0.067% methyl paraben completely inhibited growth and ochratoxin A production by both fungi. Sodium bisulfite at 0.1%, the maximum level tested, was found to inhibit growth of A. sulphureus and P. viridicatum by 45 and 89%, respectively. Toxin production was inhibited by 97 and 99%, respectively. Sodium propionate (0.64%) at pH 5.5 inhibited growth of A. sulphureus and P. viridicatum by 76 and 90%, respectively. Toxin production was inhibited by greater than 99% for each fungus. Antimicrobial agents were ranked as to effectiveness by comparing the level required for complete inhibition of ochratoxin A production to the highest antimicrobial agent level normally used in food. At pH 4.5, the most effective inhibitor of growth and toxin production was potassium sorbate, followed by sodium propionate, methyl paraben, and sodium bisulfite, respectively, for both fungi. However, at pH 5.5, the most effective antimicrobial agents for inhibiting ochratoxin production were methyl paraben and potassium sorbate, followed by sodium propionate. Sodium bisulfite was not highly inhibitory to these toxigenic fungi at the higher pH value tested.     

Inhibition by antimicrobial food additives of ochratoxin A production by Aspergillus sulphureus and Penicillium viridicatum.

The effects of antimicrobial food additives on growth and ochratoxin A production by Aspergillus sulphureus NRRL 4077 and Penicillium viridicatum NRRL 3711 were investigated. At pH 4.5, growth and toxin production by both A. sulphureus and P. viridicatum were completely inhibited by 0.02% potassium sorbate, 0.067% methyl paraben, 0.0667% methyl paraben, and 0.2% sodium propionate. At pH 5.5, 0.134% potassium sorbate and 0.067% methyl paraben completely inhibited growth and ochratoxin A production by both fungi. Sodium bisulfite at 0.1%, the maximum level tested, was found to inhibit growth of A. sulphureus and P. viridicatum by 45 and 89%, respectively. Toxin production was inhibited by 97 and 99%, respectively. Sodium propionate (0.64%) at pH 5.5 inhibited growth of A. sulphureus and P. viridicatum by 76 and 90%, respectively. Toxin production was inhibited by greater than 99% for each fungus. Antimicrobial agents were ranked as to effectiveness by comparing the level required for complete inhibition of ochratoxin A production to the highest antimicrobial agent level normally used in food. At pH 4.5, the most effective inhibitor of growth and toxin production was potassium sorbate, followed by sodium propionate, methyl paraben, and sodium bisulfite, respectively, for both fungi. However, at pH 5.5, the most effective antimicrobial agents for inhibiting ochratoxin production were methyl paraben and potassium sorbate, followed by sodium propionate. Sodium bisulfite was not highly inhibitory to these toxigenic fungi at the higher pH value tested.     

Ochratoxin-A production in Brazilian dry beans (Phaseolus vulgaris L.)

Ochratoxin A was produced, at concentrations of about 200 mg kg1 of dry beans (Phaseolus vulgaris L.) of each of five Brazilian commercial varieties. Both intact and decorticated kernels of the varieties Preto, Branco, Rosinha, Roxo and Carioca (22% moisture) were inoculated withAspergillus alutaceous and incubated at 25°C for 28 days. Results from thin-layer and column chromatography, mass, infrared, 1H-nuclear magnetic resonance and UV-spectrometry showed that 1) the common bean is a highly stimulatory substrate for the bioproduction of ochratoxin A and 2) the putative toxin extracted by the method of Soares & Rodriguez-Amaya was in fact ochratoxin A. Removal of the seed coat resulted in increased OTA production for all varieties, particularly for the Rosinha, Roxo and Carioca.     

Production of ochratoxin A by Aspergillus ochraceus NRRL-3174 before and after exposures to 60Co irradiation.

Spores from the toxigenic organism Aspergillus ochraceus NRRL-3174 were exposed to specific levels of gamma irradiation and then allowed to germinate on selected media. Increases in ochratoxin A production by irradiated, compared to non-irradiated, spores were observed after inoculation of spores onto a cracked red wheat or into a synthetic liquid medium. Variations in daily ochratoxin production were also observed for control and irradiated spore-derived cultures developing on both media, with maximum toxin production varying from 7 to 11 days of incubation. The most notable increases in ochratoxin A production occurred from cultures developing from spores having been irradiated with 10, 25, or 50 krad. Exposures to 400 or 600 krad resulted in complete inhibition of spore germination and, consequently, no ochratoxin production. Of the two substrates used, wheat and synthetic, the quantities of ochratoxin A produced were significantly lower in the synthetic media than on the natural substrate. Higher and more rapid toxin production occurred from spores having been irradiated with 10, 25, 50, and 100 krad than occurred from the non-irradiated control spores when grown on synthetic media. Cultures derived from spores having been exposed to 10, 25, 50, and 100 krad produced significantly higher levels of ochratoxin A after 8 days of incubation on natural substrate than did the controls. Analysis of variance revealed that substrate, length of incubation, as well as irradiation levels all affected the time required to produce maximum levels of ochratoxin A.     

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.     

Ochratoxin A: study of grain contamination

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 micrograms/kg. An analysis of forage grain found ochratoxin A at concentrations of 440-3250 micrograms/kg.     

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.     

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.     

Aflatoxin and Ochratoxin Production by <Emphasis Type="Italic">Aspergillus</Emphasis> Species Under Ex Vivo Conditions

Aspergillus species are increasingly important human pathogens. It is not known whether toxic metabolites of many of these pathogenic species can act as virulence factors in aspergillosis. We examined isolates of aflatoxin and ochratoxin-producing species for toxin production in ex vivo conditions. Seven of the 21 aflatoxin-producing isolates screened produced aflatoxin at 35 and 37°C on the general medium yeast extract sucrose agar (YES). However, none of them produced toxin at these temperatures on brain heart infusion agar (BHA), a medium that mimics human tissue, or on BHA with modified pH or sugar levels. Six of the 12 ochratoxin-producing isolates examined produced toxin at 35°C on YES. All three isolates of A. alliaceus produced ochratoxin on BHA or modified BHA at 37°C. One strain of A. pseudoelegans produced a minute amount of ochratoxin on modified BHA at 37°C. These data indicate that aflatoxin is an unlikely virulence, factor but that ochratoxin may be a potential virulence factor in aspergillosis.     

Metabolism of ochratoxin A by primary cultures of rat hepatocytes.

Association of ochratoxin A with cultured rat hepatocytes occurs at 4 degrees C, and the saturation level in the medium is 0.3 mM ochratoxin A, with maximal binding after 60 min. At 37 degrees C the level of cell-associated ochratoxin A increased up to 6 h and remained at 2 nmol of toxin per mg of cell protein for 30 h. With increasing concentrations of ochratoxin A, increasing amounts of the toxin accumulated in the cells; saturation occurred at a concentration of 0.3 mM. Ochratoxin A was metabolized by hepatocytes at 37 degrees. (4R)-4-Hydroxyochratoxin A appeared in the medium at a maximal level (about 30 nmol/mg of cell protein) at an ochratoxin A concentration of 0.25 mM after 48 h of incubation. Small amounts of (4S)-4-hydroxyochratoxin A were detected only after incubation for 22 h or longer.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Metabolism of aflatoxin, ochratoxin, zearalenone, and three trichothecenes by intact rumen fluid, rumen protozoa, and rumen bacteria.

The effect of rumen microbes on six mycotoxins (aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin, diacetoxyscirpenol, and deoxynivalenol ) considered to be health risks for domestic animals was investigated. The mycotoxins were incubated with intact rumen fluid or fractions of rumen protozoa and bacteria from sheep and cattle in the presence or absence of milled feed. Rumen fluid had no effect on aflatoxin B1 and deoxynivalenol . The remaining four mycotoxins were all metabolized, and protozoa were more active than bacteria. Metabolism of ochratoxin A, zearalenone, and diacetoxyscirpenol was moderately or slightly inhibited by addition of milled feed in vitro. The capacity of rumen fluid to degrade ochratoxin A decreased after feeding, but this activity was gradually restored by the next feeding time. Ochratoxin A was cleaved to ochratoxin alpha and phenylalanine; zearalenone was reduced to alpha-zearalenol and to a lesser degree to beta-zearalenol; diacetoxyscirpenol and T-2 toxin were deacetylated to monoacetoxyscirpenol and HT-2 toxin, respectively. Feeding of 5 ppm (5 mg/kg) of ochratoxin A to sheep revealed 14 ppb (14 ng/ml) of ochratoxin A and ochratoxin alpha in rumen fluid after 1 h, but neither was detected in the blood. Whether such conversions in the rumen fluid may be considered as a first line of defense against toxic compounds present in the diet is briefly discussed.     

Ochratoxin A,an in vivo inhibitor of renal phosphoenolpyruvate carboxykinase

Ochratoxin A, a nephrotoxin produced as a secondary metabolite by A. ochraceus, is a potent inhibitor of renal PEPCK activity, in vivo. When fed orally to rats for 2 days, renal PEPCK activity is reduced 50% by a total dose of 0.3-0.5 mg toxin. Renal gluconeogenic capacity is reduced only after PEPCK activity is inhibited by 50%. Hepatic PEPCK activity is unaffected up to 1.5-2.0 mg ochratoxin A, which were the highest doses tested. Other enzymes located in proximal convoluted tubules, including phosphatedependent glutaminase, γ-glutamyl transpeptidase, pyruvate carboxylase, and Na,K-ATPase, are not affected. Renal protein synthesis from [³H]phenylalanine or [³H]leucine is inhibited 30–40% by ochratoxin A in vivo. By covalently coupling the toxin to albumin with carbodiimide or mixed anhydride, the inhibitory effect on renal PEPCK activity is retained, but protein synthesis is not affected and cytological evidence of nephrotoxicity is lost. Injection of the ochratoxin A-albumin carbodiimide complex results in a decrease of hepatic PEPCK activity as well. Removal of the phenylalanine group from the toxin prevents the in vivo inhibition of PEPCK activity, as well as protein synthesis. We conclude that the decrease in renal PEPCK activity, in vivo, requires the phenylalanine group of ochratoxin A, and occurs by a mechanism independent of the known nephrotoxicity effects.     

PVP-coated graphene oxide for selective determination of ochratoxin A via quenching fluorescence of free aptamer

In this paper, we developed a simple method to detect fungi toxin (ochratoxin A) produced by Aspergillus Ochraceus and Penicillium verrucosumm, utilizing graphene oxide as quencher which can quench the fluorescence of FAM (carboxyfluorescein) attached to toxin-specific aptamer. By optimizing the experimental conditions, we obtained the detection limit of our sensing platform based on bare graphene oxide to be 1.9 μM with a linear detection range from 2 μM to 35 μM. Selectivity of this sensing platform has been carefully investigated; the results showed that this sensor specifically responded to ochratoxin A without interference from other structure analogues (N-acetyl-l-phenylalanine and warfarin) and with only limited interference from ochratoxin B. Experimental data showed that ochratoxin A as well as other structure analogues could adsorb onto the graphene oxide. As compared to the non-protected graphene oxide based biosensor, PVP-protected graphene oxide reveals much lower detection limit (21.8 nM) by two orders of magnitude under the optimized ratio of graphene oxide to PVP concentration. This sensor has also been challenged by testing 1% red wine containing buffer solution spiked with a series of concentration of ochratoxin A.     

Biosynthesis of ochratoxins by Aspergillus ochraceus.

Shaken liquid fermentation of an isolate of Aspergillus ochraceus showed growth-associated production of ochratoxins A and B, followed by production of a related polyketide diaporthin. Later, between 150 and 250 h, mellein accumulated transitorily. In contrast, shaken solid substrate (shredded wheat) fermentation over 14 days produced mainly ochratoxins A and B (ratio ca. 5:1) in very high yield (up to 10 mg/g). In these systems experiments with 14C-labelled precursors and putative intermediates revealed temporal separation of early and late stages of the ochratoxin biosynthetic pathway, but did not support an intermediary role for mellein. The pentaketide intermediate ochratoxin beta was biotransformed very efficiently into both ochratoxins A and B, 14 and 19%, respectively. The already chlorinated ochratoxin alpha was only biotransformed significantly (4.85%) into ochratoxin A, indicating that chlorination is mainly a penultimate biosynthetic step in the biosynthesis of ochratoxin A. This was supported by poor (1.5%) conversion of radiolabelled ochratoxin B into ochratoxin A. Experiments implied that some ochratoxin B may arise by dechlorination of ochratoxin A.     

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%.     

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.     

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.     

Ochratoxin A in pig blood: method of analysis and use as a tool for feed studies

A procedure is presented for screening the quality of feed in respect to ochratoxin A contamination based upon the analysis of ochratoxin A in pig blood. Representative samples from large feed lots may be obtained by using pigs as in vivo sample collectors which enrich the toxin and forms homogeneous samples in the blood. The spectrofluorometric procedure for ochratoxin A analysis (K. Hult and S. Gatenbeck, J. Assoc. Off. Anal. Chem. 59:128-129, 1976) has been adapted to pig blood and has been simplified to involve only three extraction steps. A volume of 2.5 ml of blood or plasma is needed, and the detection limit is 2 ng of ochratoxin A per ml. The disappearance of ochratoxin A from pig blood as a function of time has been studied. A feeding experiment with ochratoxin A has been performed, and the time course of the concentration of ochratoxin A in blood has been followed during the experiment.