Degradation of ochratoxin A by Aspergillus species

Mycotoxin contamination of agricultural products is a serious health hazard throughout the world. Besides attempts to eliminate mycotoxins from contaminated substrates by physical and chemical methods, the ability of microbes to degrade mycotoxins is now being widely examined. In this study, several Aspergillus species were examined for their ability to degrade ochratoxin A. A. fumigatus and black Aspergillus strains were found to detoxify ochratoxin A in culture media. The kinetics of ochratoxin A detoxification by an atoxigenic A. niger strain was examined by thin layer chromatography, high-performance liquid chromatography and an immunochemical technique. A. niger CBS 120.49 was found to effectively eliminate ochratoxin A from both liquid and solid media, and the degradation product, ochratoxin alpha, was also decomposed.     

Mycotoxins in cereal grain. Part IV. Inactivation of ochratoxin A and other mycotoxins during ammoniation

Addition of ammonia to final concentration 2% inactivates ochratoxin A, aflatoxin, citrinin, penicillic acid and partially zearalenon at temperature 20-50 °C. Detoxification of contaminated cereal grain (wheat, corn or barley) can be performed on a farm using ammoniation without special investment during 4 to 6 weeks. Ammoniation changes nutritional value of grain as feed in a small extent.     

Occurrence of the mycotoxins ochratoxin A, zearalenone and deoxynivalenol in feed components.

The mycotoxin contamination of feed components used by the Dutch cooperative feed industry was surveyed to estimate the risk for animal production losses. Of 89 randomly and 6 selectively taken samples of raw materials harvested in 1988 and 1989 27% were contaminated with ochratoxin A (OCHRA), 31% with zearalenone (ZEA) and 20% with deoxynivalenol (DON). The mean content (microgram/kg) of all positive randomly taken samples was 18 (OCHRA), 62 (ZEA) and 630 (DON). The highest level (microgram/kg) for all samples was 120 (OCHRA) in barley, 3100 (ZEA) in corn cob mix and 1900 (DON) in maizegluten feed. The results of this survey show that feed components are often contaminated with mycotoxins. However, the contamination level could only sporadically cause production losses in animal husbandry.     

Non-specificity of nutritional substrate for ochratoxin A production by isolates of Aspergillus ochraceus

Aspergillus ochraceus is an important contaminant of diverse substrates, such as cereals, coffee, grapes and derivates. This fungus produce a nephrotoxic metabolite, ochratoxin A (OTA), whose presence on food and feeds may be an important risk for animal and human health. The aim of this work was to evaluate the significance of the origin of A. ochraceus isolates on their OTA production patterns on different substrates (yeast extract sucrose (YES) broth, irradiated barley grains, irradiated green coffee beans and sterilized grapes) and under different environmental conditions. Results did not show a significant influence of the isolation source on OTA-production profiles by A. ochraceus isolates on several substrates, since the isolates which produced the highest OTA amounts in vitro (YES medium) were also the isolates with the highest OTA yields on the other substrates. Abiotic factors assayed (water activity, temperature and substrate) affected significantly OTA productions by A. ochraceus. Maximum OTA amounts were detected at 25 degrees C and 0.98 a(w) on all substrates tested. The highest OTA accumulations found on the different substrates were: green coffee beans (> 2 mg g(-1)), barley grains (approximately 1 mg g(-1)), YES medium (13.9 microg ml(-1)) and grape (approximately 3 ng g(-1)).     

Occurrence of ochratoxin A and toxigenic potential of fungal isolates from Spanish grapes

This paper reports the results from an extensive survey of filamentous fungi isolated from wine‐producing grapes and their capacity to produce ochratoxin A (OTA) on Czapek Yeast Autolysate agar (CYA), in order to assess their potential for producing this toxin in grapes. Grapes were sampled from four Spanish wine‐producing regions during 2001. The fungal infection in berries increased with time, reaching 100% at harvest. A total of 386 isolates of Aspergillus section Nigri and 10 of Aspergillus section Circumdati were isolated and tested for their ability to produce OTA in CYA. 21 strains produced OTA (18 Aspergillus section Nigri and 3 Aspergillus section Circumdati). Aspergillus section Circumdati isolates produced higher amounts of OTA than Aspergillus section Nigri ones, with means of 10.76 µg g^?1 CYA and 1.42 µg g^?1 CYA, respectively. Despite this, black aspergilli are believed to be highly responsible for the OTA levels found in musts and wines, as it is more widespread in grapes. Musts (n = 40) produced from the grapes collected were analysed. 15% were found to contain OTA, concentrations ranging from 0.091 to 0.813 ng ml^?1 (detection limit: 0.07 ng ml^?1), but no correlation was found with the ochratoxigenic moulds isolated from grapes. Copyright © 2004 Society of Chemical Industry     

Production of mycotoxins by selected Fusarium graminearum and F. crookwellense isolates.

Corn cultures (five isolates each of Fusarium graminearum Group 1 from wheat crowns, Group 2 from scabby wheat grains and from ear rot of corn and five isolates of F. crookwellense) were screened for their ability to produce deoxynivalenol (DON), nivalenol (NIV), fusarenon-x (FUS-X) and zearalenone (ZEA). Nine of the ten F. graminearum isolates from wheat produced DON (5-165 micrograms g-1) but none produced either NIV or FUS-X. Conversely, 3/5 and 2/5 of the F. graminearum isolates from corn produced NIV (5-40 micrograms g-1) and FUS-X (5-7 micrograms g-1), respectively, while none produced DON. All but one of the F. graminearum isolates produced ZEA (2-1160 micrograms g-1). None of the F. crookwellense isolates produced DON, but 5/5 and 4/5 produced NIV (6-170 micrograms g-1) and FUS-X (3-90 micrograms g-1), respectively, and all produced ZEA (605-1030 micrograms g-1). The results confirmed previous findings on the presence of two distinct F. graminearum chemotypes.     

Occurrence of mycotoxins (ochratoxin A, deoxynivalenol) and toxigenic fungi in Moroccan wheat grains: impact of ecological factors on the growth and ochratoxin A production

The aim of the present work was to evaluate the contamination of some samples, taken from Moroccan wheat grains, by ochratoxin A (OTA), deoxynivalenol (DON) and the associated toxigenic fungi. Moreover, we focused on the influence of environmental factors on both the growth and OTA production by three strains of Aspergillus. The results showed that only few samples were contaminated by the two mycotoxins (2 samples for OTA and 7 for DON). The main isolated fungi belong to the Aspergillus, Penicillium and Fusarium genus; 74 Aspergillus and 28 Penicillium isolates were tested for their ability to produce OTA. Only 2 A. alliaceus and 14 A. niger were able to synthesize OTA. However, none of Penicillium isolates can produce this toxin under the conditions mentioned. In respect of the effects of the temperature and water activity (aw), the optimal conditions for the growth and OTA production were different. While the optimal conditions of growth for A. alliaceus and A. terreus are 30 degrees C and 0.98 aw, A. niger preferred 0.93-0.95 aw at 25 degrees C, whereas the optimal production of OTA was observed at 30 degrees C for both A. alliaceus and A. niger at 0.93 and 0.99 aw, respectively.     

A simple HPLC method for the determination of the mycotoxins ochratoxin A and B in blood serum of swine.

This paper presents a simple method for the determination of ochratoxins A (OTA) and B (OTB) in pig blood serum. The method includes serum acidification (pH < 1.6) and precipitation of protein with 15% trichloroacetic acid, liquid partitioning with dichloromethane and fluorescence detection. The estimated detection limits were 0.1 ng OTA/ml and 0.2 ng OTB/ml. The mean recoveries from artificially contaminated samples (n = 6 replicates/mycotoxin) spiked at 0.3, 1 and 3ng OTA and OTB/ml, respectively, were 86.8% (s.d. = 8.4) for OTA and 90.0% (s.d. = 9.8) for OTB. Forty-nine Romanian pig blood serum samples (94% of 52 analysed) were found to be naturally contaminated with OTA in the range 0.1-13.4 ng/ml. No sample was found positive for OTB. The method is technically simple, specific, cost effective, suitable for large sample throughput and requires small amount of sample and reagents. It fulfils the criteria for a routine method and could be a suitable toolfor surveying OTA in pig herds and in slaughtered pigs.     

Occurrence of ochratoxin A in sweet wines produced in Spain and other countries

A survey for the presence of ochratoxin A (OTA) was undertaken from 2001 to 2005 in 188 samples of sweet wines produced in Spain and in 102 samples originating from other countries: France (n = 49), Austria (9), Chile (9), Portugal (9), Greece (6), Italy (5), Germany (3), Hungary (2), Slovenia (2), Switzerland (2), Canada (1), Japan (1), New Zealand (1), Ukraine (1), South Africa (1) and the USA (1). The analytical method was based on immunoaffinity chromatography clean-up and high-performance liquid chromatography (HPLC) with fluorescence detection. The limit of detection (defined as a signal-noise ratio = 3) was estimated to be 0.01 µg l^-1. The limit of quantification (0.02 µg l^-1) was checked as being the lowest measurable concentration. OTA was detected in 281 out of 290 samples analysed (96.9% positive) at concentrations ranging from 0.01 to 4.63 µg l^-1. The overall mean and median levels were estimated to be 0.50 and 0.14 µg l^-1, respectively. In Spanish sweet wines OTA was found in 99% of the samples, with mean and median values of 0.65 and 0.19 µg l^-1, respectively. The mean value obtained in this study for OTA in the Spanish sweet wines would result in an intake of about 37.5 and 3.2 ng day^-1 of OTA for regular consumers and for the overall population, respectively. These figures represent a minor contribution to the provisional tolerable weekly intake (PTWI) or TWI established by the Joint Expert Committee on Food Additives (JECFA) and the European Food Safety Authority: 3.8 and 3.1% for regular consumers; and 0.4 and 0.3% for the whole adult population, respectively.     

Long term administration of low doses of mycotoxins in poultry. 2. Residues of ochratoxin A and aflatoxins in broilers and laying hens after combined administration of ochratoxin A and aflatoxin B1

The effects of combined administration of ochratoxin A (OA) and aflatoxin B1 (AFB1) on the occurrence and the levels of residues of mycotoxins in poultry have been investigated. Male broilers and laying hens were fed from 14 days old with standard diets contaminated with 50 micrograms/kg OA and 50 micrograms/kg AFB1. Two groups of broilers and hens were withdrawn from contaminated feed at 37 and 88 days, respectively. At the time of sacrifice no significant lesions were found. Residues were compared with those found after administration of either toxin alone in former trials. Combined treatment resulted in higher content of OA in broiler livers (40 versus 5.0 micrograms/kg) and, to a lesser extent, in kidneys and skin, and of AFB1 in broiler liver and kidney (0.15 versus 0.02 microgram/kg and 0.40 versus 0.05 microgram/kg respectively). Laying hens showed smaller differences (0.20 versus 0.10 microgram/kg in liver and 0.32 versus 0.08 in kidneys). Withdrawal from treatment led to the almost complete disappearance of OA residues in broilers and in hens. These results show a synergistic effect of OA and AFB1, particularly in broilers.     

Effect of Capsicum carotenoids on growth and ochratoxin A production by chilli and paprika Aspergillus spp. isolates

The aim of this study was to determine the effect of a natural carotenoid mixture (Capsantal FS-30-NT), containing capsanthin and capsorubin, on growth and mycotoxin production of ochratoxin A-producing A. ochraceus, A. westerdijkiae, and A. tubingensis isolates.One isolate of each species, previously isolated from paprika or chilli, was inoculated on Czapek Yeast extract Agar (CYA) medium supplemented with different amounts of capsantal (0 to 1%) and incubated at 10, 15 and 25 °C for 21 days. Growth rates and lag phases were obtained, and OTA production was determined at 7, 14 and 21 days.The taxonomically related A. ochraceus and A. westerdijkiae showed the same behavior at 15 °C, but A. ochraceus was able to grow at 10 °C and had higher growth rates at 25 °C. A. tubingensis had the highest growth rates and lowest OTA production capacity of the assayed isolates, and it was not able to grow at 10 °C.Capsantal addition resulted in increased lag phases at 15 °C for all the strains, while growth rates remained rather constant. At 25 °C capsantal reduced growth rates, with rather constant lag phases. However, the effect of capsantal on OTA production was inconclusive, because it depended on temperature or time, and mostly was not significant.Low temperature has been a crucial factor in OTA production, regardless of the capsantal concentration tested, especially for A. tubingensis and A. westerdijkiae. Industrial storage temperature for paprika and chilli is approximately 10 °C. If this temperature is maintained, mould growth and OTA production should be reduced.     

Effect of water activity and temperature on mycelial growth and ochratoxin A production by isolates of Aspergillus ochraceus on irradiated green coffee beans

Aspergillus ochraceus as a fungal contaminant and ochratoxin A (OTA) producer plays an important role in coffee quality. Temperature and water activity (a(w)) significantly influence mycelial growth and OTA production by isolates of A. ochraceus on green coffee beans. Maximum mycelial growth was found at 30 degrees C and 0.95 to 0.99 a(w). A marked decrease in growth rate was observed when temperature and a(w) were reduced. At 0.80 a(w), mycelial growth occurred only at 30 and 20 degrees C for one isolate. Maximum OTA production was found at 20 degrees C and 0.99 a(w). At 10 degrees C, OTA was not produced, regardless of a(w). Similarly, no OTA was detected at 0.80 a(w). OTA production ranged from the limit of detection (40 ng g(-1) of green coffee) to 17,000 ng g(-1) of green coffee. Significant intraspecific differences in mycelial growth and OTA production were found. Primary data for lag phases prior to mycelial growth under the influence of temperature and a(w) were modelled by multiple linear regression, and the response surface plots were obtained.     

Fusarium mycotoxins and ochratoxin A in cereals and cereal products: results from the Bavarian Health and Food Safety Authority in 2004

Results from the Bavarian Health and Food Safety Authority on contamination of cereals and cereal products from the Bavarian market with the mycotoxins deoxynivalenol (DON), zearalenone (ZEA), and ochratoxin A (OTA) and of maize meal and semolina with fumonisins (FUM) in the year 2004 are presented. Contamination rates and levels of DON, ZEA, and OTA were low and did not exceed the maximum levels. However, a 92% contamination rate and high levels of FUM in maize meal and semolina were measured. Contamination levels of mycotoxins are discussed and evaluated with respect to possible health implications for consumers.     

高效液相色谱-荧光检测法检测大米中的赭曲霉毒素A

利用高效液相色谱-荧光检测法测定大米中的赭曲霉毒素A含量,确定了检测条件为：Agilent C18 (150 mm×4.6 mm,5 μm)为色谱柱,柱温25 ℃,流动相为乙腈-水（1%甲酸）= 45∶55,流速1.0 mL/min,进样量10 μL;激发波长333 nm,发射波长460 nm。结果表明,赭曲霉毒素A在0～200 ng/mL范围内线性关系良好（R2= 0.999 16）,平均加样回收率为77.7%～81.1%,平均相对标准偏差（RSD）为2.1%～3.1%,检出限为0.8 ng/kg。该方法快速准确,适用于大米中的赭曲霉毒素A含量的测定。     

A polyphasic approach to the identification of ochratoxin A-producing black Aspergillus isolates from vineyards in Sicily

Aspergillus strains belonging to section Nigri isolated during a two year survey in eight Sicilian vineyards located on the slopes of Mount Etna (Sicily, Italy) were analysed analyzed in order to characterize species responsible for ochratoxin A (OTA) contamination of grapes. The polyphasic approach permitted analysis of biodiversity of Aspergillus isolates in relation to their morphology, ochratoxigenicity and genetic variability. We assessed OTA production by A. carbonarius, A. niger, A. tubingensis and A. japonicus using an enzyme-linked immunosorbent assay. A. carbonarius isolates were the strongest OTA producers. A subset of 66 representative strains was selected for further DNA-based characterization. PCR assays using species-specific primers discriminated between A. niger, A. carbonarius and A. japonicus on the basis of the target sequences for each species. The PCR-based methods matched morphological characterization in identifying all the black aspergilli (BA) isolates tested, whereas RFLP analysis with RsaI of isolates positive to PCRs with A. niger specific primers identified three A. tubingensis isolates. The identification of thirteen isolates was further confirmed by ITS analysis. By this method, each of the isolates was identified and assigned to an Aspergillus species. The fAFLP analysis of 40 isolates highlighted the power of this technique to discriminate different species and single strains, to verify the presence of mixed populations in the same vineyard, through homogeneous species clusters. No correlation was observed between the clusters and OTA production level or origin.     

Cholestyramine protection against ochratoxin A toxicity: role of ochratoxin A sorption by the resin and bile acid enterohepatic circulation

We have shown that the addition of cholestyramine (CHA, a resin known to bind bile salts in the gastrointestinal tract) to ochratoxin A (OTA)-contaminated rat diets reduced plasma levels of the toxin and prevented OTA-induced nephrotoxicity. To elucidate the mechanism of action of CHA, we carried out in vitro experiments to determine whether the resin may bind the toxin. For comparative purposes, binding of bile salts to the resin was also examined. Results showed that CHA binds both OTA and bile salts (taurodeoxycholate [TDC] and taurocholate [TCA]). Also, CHA showed greater affinity for OTA and TDC than for TCA. At 1 mM concentration, 96% of OTA and 80% of TDC were bound to the resin, while for TCA binding was only 50%. However, saturation of the resin was reached at higher levels with bile acids compared to OTA (3.67 mmol/g resin for TCA and 3.71 mmol/g resin for TDC versus 2.85 mmol/g resin for OTA). To characterize the nature of the binding of the toxin to CHA, NaCl (0 to 200 mM) was added to a fixed amount of OTA or bile acids. As expected, TCA absorption was decreased by the addition of NaCl (<50 mM), indicating electrostatic binding. However, OTA and TDC sorption was decreased only at high concentrations of NaCl (>150 mM), suggesting a stronger binding to the resin than that shown with TCA. Sequential competitive studies demonstrated that CHA binds more OTA than TCA. The results of the in vivo study show the role of bile salts in OTA absorption. The toxin's plasma levels at 1 and 3 h after a single oral dose of OTA were significantly decreased in bile salt-depleted rats compared to the control. Thus, the alteration of the bile salt biliary pool and OTA enterohepatic circulation may be an additional mechanism of action of the resin against mycotoxin toxicity.     

Environmental factors and weak organic acid interactions have differential effects on control of growth and ochratoxin A production by Penicillium verrucosum isolates in bread

Studies were conducted to examine the effect of water activity (a(w), 0.97, 0.95, 0.93), pH (4.5, 6.0) and preservative type and concentration (potassium sorbate (KS), calcium propionate (CaP), 300 and 3000 ppm) on colonisation and ochratoxin A (OTA) production by three strains of Penicillium verrucosum on bread analogues. At 25 degrees C, colony extension from a point source was similar for all three strains at the different environmental treatments examined, with optimum growth at 0.97 and 0.96 a(w) and pH 6. Temporal OTA production over 36 days varied significantly with strain, pH and a(w) level. Maximum OTA was produced at approximately 28-36 days for two of the strains with an optimum at 0.93 a(w) at pH 6.0. One strain produced 75-80 microg g(-1) analogue under these conditions. However, at pH 4.5, optimum OTA was produced at 0.95 a(w) after a similar time period on bread analogues, with maximum amounts of about 25-35 microg g(-1) bread. KS and CaP incorporation inhibited growth completely at pH 4.5 and 3000 ppm at all three a(w) levels. However, at suboptimal concentration, little growth inhibition occurred. At pH 6, even with 3000 ppm, growth of all three strains occurred. Where effective inhibition of growth was achieved (3000 ppm/pH 4.5, all a(w) levels), no OTA was detected. However, when 300 ppm was used, up to 7 microg g(-1) was detected in bread analogues at pH 4.5. At pH 6, OTA was produced at all treatment conditions by all three strains, although preservative-treated bread had less present. Statistical analysis showed that many of the one-, two- and three-way interactions had a significant effect on growth and OTA production. These results suggest that there is a significant risk from OTA contamination if lower concentrations of existing preservatives are used in wheat-based bakery products contaminated by spoilage mycotoxigenic moulds such as P. verrucosum.     

Effect of temperature and water activity on growth and ochratoxin A production by Australian Aspergillus carbonarius and A. niger isolates on a simulated grape juice medium

The effect of water activity (0.92, 0.95, 0.965 and 0.98) and temperature (15 °C, 25 °C, 30 °C and 35 °C) on growth rate and ochratoxin A (OA) production by five strains of Aspergillus carbonarius and two strains of A. niger isolated from Australian vineyards was characterised on a synthetic grape juice medium. Maximum growth for A. carbonarius occurred at ca 0.965 a_w and 30 °C, and for A. niger, at ca 0.98 a_w and 35 °C. The optimum temperature for OA production was 15 °C and little was produced above 25 °C. The optimum a_w for toxin production was 0.95–0.98 for A. carbonarius and 0.95 for A. niger. Toxin was produced in young colonies after and, typically, did not continue to accumulate the entire surface area of the plate was colonised. Rather, the amount decreased as colonies aged. Trends for growth and OA production were similar among Australian isolates and those from European grapes, as reported in the literature.