Influence of carbon and nitrogen sources on ochratoxin A production by two species of Penicillium isolated from poultry feeds

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural commodities in the field and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several species of Aspergillus and Penicillium. In this study, the influence of carbon and nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra Pradesh, India. The isolated ochratoxigenic Penicillium species were identified, screened and characterised as OTA producers by high performance thin layer chromatography (HPTLC) and confirmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate (CYA) medium with different carbon (C) and nitrogen (N) sources at pH 6.5 and incubated at 25 ± 2°C under dark condition. Maximum OTA production was recorded in the presence of D-glucose followed by D-galactose and D-lactose as carbon sources. Similarly, the maximum amount of OTA production was observed in thiourea followed by potassium nitrate as nitrogen source. However, OTA production, final pH of the medium, and mycelial yield and OTA production of both the species of Penicillium varied with C and N present in the medium. The kinetics of the both species of Penicillium was observed for 30 days at an interval of three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively.     

Effect of indigenous mycobiota on ochratoxin A production by Aspergillus carbonarius isolated from soil

This study determined the biotic interaction between 30 non-toxigenic indigenous strains of Aspergillus niger aggregate, Aspergillus flavus, Trichoderma spp., Mucor spp., Cladosporium spp., Ulocladium spp., Curvularia spp., Absidia spp., Geotrichum spp. and Acremonium spp., isolated from soil destined for maize crops, with respect to their ability to prevent ochratoxin A (OTA) production by A. carbonarius on “in vitro” assay, on liquid and solid medium. OTA production was completely inhibited when A. carbonarius was inoculated in a interactive mixed culture with all A. niger aggregate strains assayed, a 80 % of Trichoderma spp. strains, a 40 % of Cladosporium spp. strains, Acremonium spp and Geotrichum spp; only one strain of A. flavus tested was able to completely inhibit the mycotoxin accumulation. OTA production increased when A. carbonarius ACS 8 was growing on liquid interactive mixed culture with Mucor spp strains. These results demonstrated that OTA production by Aspergillua carbonarius strain was significantly influenced by the presence of different non-toxigenic fungal strains when growing together on paired cultures.     

Allium cepa as a biomonitor of ochratoxin A toxicity and genotoxicity

Ochratoxin A (OTA) is a toxin produced by Aspergillus and Penicillum moulds. Since OTA has not yet been evaluated in plant systems, this paper focused on describing the controversial effect OTA in an Allium root test model, which has known sensitivity to genotoxins and could be useful in toxin screening. Analyses of root growth and the root meristematic zone in response to OTA treatment were undertaken. The results show OTA toxicity to root growth at a concentration of 10 ug·ml^?1 associated with inhibition of proliferation activity. Cytological changes observed in the Allium chromosome aberrations assay, at a concentration of 5.0 ug·ml^?1, showed that OTA was able to induce genotoxicity at the chromosome level. These results indicate that plants cells (Allium cepa) are very sensitive to the mycotoxin OTA, as observed at the highest concentration. Under these conditions, OTA produced toxicity and cytogenetic injury. Evidence in vitro and in vivo indicates that OTA can induce damage at the DNA level.     

New Insight into the Ochratoxin A Biosynthetic Pathway through Deletion of a Nonribosomal Peptide Synthetase Gene in Aspergillus carbonarius

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine, and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been elucidated in Penicillium species. In Aspergillus species, only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase (NRPS) in OTA-producing A. carbonarius ITEM 5010 has eliminated the ability of this fungus to produce OTA. This is the first report on the involvement of an nrps gene product in OTA biosynthetic pathway in an Aspergillus species. The absence of OTA and ochratoxin α, the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β, the dechloro analog of ochratoxin α, were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight into the biosynthetic pathway of the toxin.     

Modulation of ochratoxin A induced DNA-damage in urothelial cell cultures

Despite good evidence for a genotoxic potential of ochratoxin A (OTA), the mechanism of OTA-induced genotoxicity (direct or indirect?) is still unclear. This calls for a further characterization of OTA-related DNA damage, and investigations of factors that may modulate dose-effect relationships in cells. Since bladder epithelium is a target tissue for the toxicity of OTA, its effects were studied in cultures of human bladder carcinoma (H5637) cells. Cytotoxicity of OTA, assessed by Neutral red (NR) uptake or Alamar-Blue assay, is concentration- and time-dependent: Upon 24 h treatment of 5637 cells, NR uptake is reduced by 50% with OTA concentrations of ≥0.2 microM, but not with 3 h treatment of the cells. Since cytotoxicity of OTA was not affected by addition of xenobiotic metabolizing enzymes (S-9 mix), it appears to be unrelated to biotransformation of the mycotoxin. Also, addition of S-9 mix did not significantly affect the genotoxicity of OTA as studied by alkaline single cell gel electrophoresis (Comet assay). DNA damage was detectable after 3 h treatment of cells at OTA concentrations between 0.1 and 1 microM, and increased further at higher concentrations. The magnitude of OTA-induced DNA damage did not increase with longer treatment times (18, 24 h), probably due to repair processes in the cells. Repair of OTA-induced lesions is quite efficient in kidney (Arch Toxicol 2002, 75, 734–741) and in porcine bladder cells (Föllmann and Lebrun, 2005, Mycotoxin Research, this volume). Interestingly, the genotoxicity of OTA is modulated by the pH of the culture medium, with higher damage at pH 5 compared to pH 7.5. In line with this, uptake studies with tritiated OTA show a higher cellular accumulation of the mycotoxin at pH 5 than in buffer of pH 7.5. Thus, bladder cells exposed to OTA in slightly acidic urine (which facilitates reabsorption) may be at higher risk.     

Effect of ochratoxin A and ochratoxin C on the monocyte and lymphocyte function

The effect of practically relevant mycotoxin concentrations on functions of immune cells was studied in in vitro experiments. Porcine mononuclear cells were exposed to a crudeAspergillus-ochraceus toxin containing OTA, a HPLC fraction identical with OTC derived from the crude toxin (RE2), as well as pure OTA and OTC in a concentration range from 0.46 to 3000 ng/ml. The influence of mycotoxin exposure on metabolic activity, mitogen induced proliferation, expression of the activation marker CD25 and the cell cycle of lymphocytes and on the formation of free oxygen radicals as well as the production of the cytokines IL-6 and TNF-α by monocytes was determined. Exposure to high concentrations of all mycotoxin preparations lead to non-specific suppression of the immune cell functions, which was related to cytotoxic effects. Low concentrations caused ambivalent reactions, especially on monocyte function. In general, the HPLC fraction RE2 had an up to 100-fold stronger effect than pure OTA. Ochratoxin-induced suppression of lymphocyte proliferation was not abrogated by phenylalanine or aspartame. The results indicate that immunomodulation can be caused by very low mycotoxin concentrations which are not related to clinical symptoms or loss of performance.     

Bee pollen, a substrate that stimulates ochratoxin A production by Aspergillus ochraceus Wilh

The capacity of bee pollen as a substrate for production of ochratoxin A (OTA) by a strain of Aspergillus ochraceus was studied. For control purposes corn, wheat and rice grains, and eleven liquid media were assayed. They were Yeast Extract Sucrose broth (YES), YES supplemented with 0.05, 0.1, 0.5, 1 and 5% bee pollen, YES supplemented with 0.5% peptone, 50% must, Wickerham medium, Aflatoxin Production medium and Coconut Broth Medium. Cultures were maintained at 28 degrees C for 4 weeks and were analyzed every seven days for OTA by liquid chromatography with fluorescence detection. OTA production in bee pollen was significantly (P < 0.01) higher than production in corn, wheat and rice grains regardless of incubation time. With regard to liquid cultures, OTA accumulation in YES supplemented with 5% bee pollen was significantly higher than in pollen-free liquid cultures. A positive correlation between the proportion of pollen added to YES medium and OTA level was observed. This is the first report concerning the use of bee pollen as a substrate to stimulate OTA production. On the basis of the preliminary results obtained in this study it can be hypothesized that bee pollen may constitute an important risk factor concerning the presence of OTA in the diet of consumers of that nutritious food.     

Aptamer-based depletion of small molecular contaminants: A case study using ochratoxin A

Based on the increasing demand for detection and depletion of small molecules like mycotoxins or pesticides in food, water, or pharmaceuticals, aptamers are gaining more importance as sensitive, specific-depletion molecules. Here, we present an aptamer-based method for depletion of ochratoxin A (OTA) as a model system and show the advantages and the limitations of aptamers in the depletion of small molecular contaminants. OTA is a mycotoxin produced by various Penicillium and Aspergillus strains and is often found in grain and grain derivatives. We immobilized a well-described DNA aptamer against OTA on an agarose gel and used the column as a clean-up system. The aptamer shows a high specificity and sensitivity for OTA: Ochratoxin B, a molecule similar to OTA, was not bound by the aptamer; and a control oligonucleotide was not able to bind OTA. After optimizing the process for better economic feasibility, the column could be used for several times without loss of aptamer activity. We investigated the location of immobilized aptamer within the gel using fluorescent-labeled aptamers. Furthermore, beer samples spiked with OTA were used to investigate aptamer activity in complex samples. Using these complex samples we have observed a significant loss of aptamer activity. We have further investigated this limitation by performing microscale thermophoresis experiments to determine the KD values of the aptamer in different complex samples like beer, coffee, juice and wine. Our results indicate that the applicability of aptamers to real processes is currently restricted by the selection buffer used during its selection process (SELEX). We therefore suggest using conditions closer to those of the later application of the aptamer during future SELEX experiments.     

Evaluation of aflatoxin B1 and ochratoxin A in interacting mixed cultures of Aspergillus sections Flavi and Nigri on peanut grains

The influence of inoculum size on the colony-forming units, production of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was determined when Aspergillus flavus and A. niger aggregate strains were cultured alone and in pairs on irradiated peanut grains at 28°C and 0.97 water activity (a_W). The results showed a marked influence of inoculum factor on fungal counts, AFB₁ and OTA production in single and paired cultures. Fungal counts of the A. niger aggregate strain in interacting cultures at 7, 14 and 21?days of incubation were significantly higher than those observed in the A. flavus strain, except in the mixed culture with 10² spores/ml of both strains. In all mixed culture assays, the AFB₁ production was significantly reduced in comparison with the accumulation of mycotoxin in single cultures. A total inhibition in AFB₁ production was observed in some interactions as 10² spores/ml of A. flavus and 10³ spores/ml of A. niger aggregate strain at 7 and 14?days, among others. With regard to OTA production, a stimulation in the interacting cultures was observed at all inoculum sizes and incubation period. The highest levels of OTA accumulation were observed at 14?days for all interacting cultures. The maximum level was reach in the culture 10³ spores/ml of A. niger aggregate and 10⁴ spores/ml of A. flavus (p?<?0.001). These results suggest that, under optimal environmental conditions in peanut grains, the interaction between A. flavus and A. niger aggregate strains could result in an inhibition of AFB₁ and in a stimulation of OTA production.     

Ochratoxin A induces apoptosis in neuronal cells

The mycotoxin ochratoxin A (OTA), which is produced by Aspergillus and Penicillium subspecies, is a frequently present contaminant of food and feedstuffs. OTA exhibits a wide range of toxic activities including nephro- and hepatotoxicity. However, little is known regarding potential neurotoxic effects of OTA. In the present study primary neurons as well as SH-SY5Y neuronal cells were incubated with increasing concentrations of OTA (0.1–2.5 μmol/L). OTA treatment resulted in a dose-dependent increase in cytotoxicity in both neuronal cell types. Caspase-9 and caspase-3 were activated in response to OTA treatment. Furthermore, caspase inhibitors were effective in partly counteracting OTA induced neurocytotoxicity. OTA induced apoptosis was accompanied by a loss of mitochondria membrane potential. Overall, present data indicated that OTA is neurotoxic at relatively low concentrations. OTA induced neurotoxicity seems to be, at least party, mediated by apoptosis. OTA may contribute to the pathogenesis of neurodegenerative diseases (e.g. Alzheimer’s and Parkinson’s disease) in which apoptotic processes are centrally involved.     

Monitoring of ochratoxin A and ochratoxin-producing fungi in traditional salami manufactured in Northern Italy

Fungi have a crucial role in the correct maturation of salami, but special attention should be addressed to the production of the nephrotoxic, immunotoxic, and carcinogenic mycotoxin ochratoxin A (OTA). In a monitoring study conducted in Northern Italy, OTA was detected by liquid chromatography coupled with mass spectrometry in 13 out 133 samples of traditional salami (9.8% of the total count). Mycological analysis of these samples yielded 247 fungal isolates which were identified to species level. The most frequent species were Penicillium nalgiovense, P. solitum, and P. chrysogenum. P. nordicum, an OTA-producing species commonly found in proteinaceous food, was not found in these samples. Three isolates were found to be Aspergillus westerdijkiae, an OTA-producing species. In order to check the results of the microbiological identification, 19 different strains of Aspergillus and 94 of Penicillium were tested for the presence of a sequence common to OTA-producing fungi by real-time PCR. None of the studied isolates, including the three A. westerdijkiae, possessed the otanpsPN target which is common to OTA-producing strains. Two out of three isolates of the A. westerdijkiae were also PCR-negative for the otanpsPN gene and did not produce OTA in culture. Conversely, this target sequence was amplified from the DNA purified from 14 salami casings including three casings harboring A. westerdijkiae. The amplification of sequences specific for OTA-producing strains performed on total genomic DNA extracted directly from salami casings provided a more suitable approach than PCR analysis of isolates from salami for the OTA-related otanpsPN gene to evaluate the risk of OTA contamination.     

Mycotoxin production by different ochratoxigenic <Emphasis Type="Italic">Aspergillus</Emphasis> and <Emphasis Type="Italic">Penicillium</Emphasis> species on coffee- and wheat-based media

Ochratoxin A (OTA) is one of the most widespread mycotoxins, and is produced by several Aspergillus or Penicillium species. Human exposure to OTA is mainly by intake of contaminated food, with cereal products, followed by coffee and red wine as the main sources of OTA. In this study, the OTA production of four ochratoxigenic fungi (two Aspergillus and two Penicillium species) was investigated in four different media, i.e. wheat and coffee model media as food-based media and two standard laboratory media (malt extract glucose agar, MEA and yeast extract sucrose agar, YES). Colony growth was documented and OTA concentrations in cultures were determined at day 2, 4 and 8 of incubation at 25°C by high-performance thin-layer chromatography (HPTLC) and high-performance liquid chromatography (HPLC). OTA production clearly depended upon time of incubation, fungal species, and medium composition. On coffee based medium, moderate OTA levels were produced by A. ochraceus BFE635 (9.8 μg/g) and by A. niger BFE632 (10.6 μg/g) on day 8 of incubation. In wheat-based medium, these strains produced much more OTA than in coffee. The highest OTA concentration (83.8 μg/g on day 8) was formed by A. ochraceus BFE635 followed by the other Aspergillus niger BFE632 (49 μg/g). Lower OTA levels were produced by P. verrucosum BFE550 and P. nordicum BFE487, in both wheat and in YES medium, whilst OTA was hardly detectable in coffee and in MEA in case of P. nordicum. Colony growth of the tested strains on different media was not indicative of OTA production. Guttation droplets developed on wheat-based medium with the Aspergillus strains within a week, and this phenomenon coincided with the high OTA amounts formed by these species. Results from this study add to our knowledge on the behaviour of ochratoxigenic fungal species when cultured on food based media.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

Effects of ochratoxin A on membrane phospholipids of the intestine of broiler chickens,practical consequences

Ochratoxin A (OTA) is a mycotoxin produced by various species of Aspergillus and Penicillium. Ochratoxin A was classified as a group 2B carcinogen and is one of the major intestinal pathogenic mycotoxins. One of the most frequent modes of intoxication is consumption of contaminated food with mycotoxins. Feed represents the major cost and has a direct impact on the economical viability of broiler’s production system, since it must contain the necessary elements that allow the animal to express the maximum genetic potential while providing its nutritional requirements. Thus, the animal has to digest the feed and absorb its nutrients, which is in direct correlation with the gastrointestinal tract, especially the small intestine and the development of the mucosal surface area. Once ingested, OTA is absorbed by passive diffusion, mainly the jejunum. Ochratoxin A’s presence affects lipid membranes and could lead to the degradation of their normal structure and functionality. All of these effects contribute to the development of malabsorption. It was very interesting to study the effect of OTA on the layer of phospholipids of the bowel. The experimental group received OTA (0.05 to mg/kg BW) through an intra-peritoneal injection, every other day for 21 days. We noted that feed conversion ratio and average daily gain were reduced. Histological studies showed important alterations at the level of the mucosal membrane of the intestine (villosities, crypts) following intra-peritoneal administration of the mycotoxin. Thinning and enlargement at the base of the villosities, hyperplasia and crypts in irregular forms, blunting and denudation were observed through the examination of intestinal morphology. Biochemical studies, such as total lipid and phospholipid compositions, allowed us to have more detailed results. All identified mucosal phospholipids were modified, particularly the phosphatidylcholine (PC) and the phosphatidylethanolamine (PE) in the jejunum mucosa. In fact, there was a decrease by 55.81% for PC, 56.66% for PE, while a significant increase by 32.91% was noted for phosphatidylserine in the jejunum. It was very interesting to study the effect of OTA on the phospholipids layer of the bowel, as the mucous membrane of the small intestine represents the main site of absorption and transformation of nutriments. To avoid such disturbances and prevent the effects of the OTA, precautions must be taken to inhibit mold growth at the level of the feed manufactory units. Phosphatidylcholine and PE administrations may represent an option that could allow reestablishment of phospholipid equilibrium in the intestine.     

Survey of mycobiota,black <Emphasis Type="Italic">Aspergillus</Emphasis> and ochratoxin A occurrence on Brazilian wine grapes

The southern state of Brazil is responsible for 90 % of the wine produced in the country; however, the mycobiota and the occurrence of ochratoxin A (OTA) on wine grapes produced in this region has never been fully characterized. To evaluate the fungal and OTA contamination in wine grapes of two varieties cultivated in southern Brazil, a survey was conducted in three regions with slightly different climates. From each region, samples were obtained at early ripeness and at harvest stage. Eight genera were isolated; Alternaria and Trichoderma were the most prevalent. Within the Aspergillus genus, the section Nigri was predominant. One uniseriate black Aspergillus was able to produce OTA under the conditions tested but no detectable levels of OTA were found in the grape samples. The low overall incidence of black Aspergillus and the absence of OTA on the grapes may be explained by the meteorological conditions at the analyzed regions, and characterizes a high quality of this product regarding mycotoxigenic contamination. This is the first study to fully describe the mycological biota of two wine grape varieties produced at three different regions of Rio Grande do Sul state.     

Exposure of neonates to ochratoxin A: first biomonitoring results in human milk (colostrum) from Chile

The mycotoxin ochratoxin A (OTA) and its metabolite ochratoxin alpha (OTα) were determined in milk and blood from nine lactating women who provided samples soon after delivery at a hospital in southern Chile. The analytical method applied liquid–liquid extraction with chloroform, and in the case of blood, an extra purification with solid phase extraction prior to HPLC analysis with fluorescence detection. OTA was detected in all human milk samples, with an average concentration of 106?±?45 ng/L (range 44–184 ng/L). Levels of OTα were 40?±?30 ng/L (LOQ 40 ng/L), but increased considerably upon enzymatic hydrolysis with ß-glucuronidase/sulfatase (up to 840?±?256 ng/L) in human milk. By contrast, there was no evidence for conjugates of OTA. The data on OTA in breast milk and levels reported in blood from women in Chile are indicative of an efficient lactational transfer of the mycotoxin. Infant exposure to OTA was estimated by considering their daily OTA intake with human milk at early stages of nursing. For the majority of milk samples, the calculated OTA intake of infants exceeded the tolerable daily intake (TDI) of 5 ng/kg body weight (bw)/day proposed by the Nordic Expert Group, and infant exposure approached the provisional tolerable doses of 14–16 ng/kg bw/day suggested by the Joint FAO/WHO Expert Committee on Food Additives (JEFCA) and by EFSA for adults. The present study documents and confirms the presence of OTA in human milk at levels where the TDI can be exceeded. These results point out the need to continue food and biological monitoring and to develop strategies, e.g. dietary recommendations to pregnant and lactating women, aimed to reduce OTA exposure in early periods of life.     

Water, temperature and gas composition interactions affect growth and ochratoxin A production by isolates of Penicillium verrucosum on wheat grain

AIMS: To examine the effect of interactions between water, temperature and gas composition on growth and ochratoxin A (OTA) production by isolates of Penicillium verrucosum in vitro and in situ on grain-based media and wheat grain. METHODS AND RESULTS: Three isolates of P. verrucosum were examined in relation to radial growth rate and OTA production, and to interacting conditions of water activity (a(w)), temperature and gas composition on a milled wheat medium. Subsequently, detailed temporal studies were carried out on gamma irradiated wheat grain over the range 0.75-0.995 a(w), 10-25 degrees C and air, 25 or 50% CO(2). This showed that optimum growth of P. verrucosum was at 0.98 a(w) in vitro at 25 degrees C, but at 0.95 a(w) and 25 degrees C on wheat grain. The a(w) minimum for growth was about 0.80 a(w), although no OTA was produced under this condition even after 56 days. Significant inhibition of growth and OTA production occurred with 50% CO(2), and 0.90-0.995 a(w) at 25 degrees C. CONCLUSIONS: The optimum and marginal conditions for growth and OTA production on wheat grain have been identified. At least 50% CO(2) is needed to inhibit growth and OTA production by >75% in moist grain (0.90-0.995 a(w)). SIGNIFICANCE AND IMPACT OF THE STUDY: First detailed identification of optimal and marginal interacting conditions of water/temperature and gas composition on growth and OTA production by P. verrucosum on wheat grain. This is a critical component of the postharvest management strategy for minimizing contamination by this important mycotoxin and predicting risk, based on environmental conditions, during drying and storage.     

Isolation and identification of ochratoxin A-producing Aspergillus section Nigri strains from California raisins

Aims: To determine incidence and levels of ochratoxin A (OTA) in California raisins and to isolate and characterize OTA‐producing fungi from California raisin vineyard populations. Methods and Results: Forty raisin clusters sampled from four California vineyards in the San Joaquin Valley were analysed for OTA content using immunoaffinity and HPLC methods. OTA was detected in 93% of the samples, at levels from 0·06 to 11·4 ng g^?1. From these raisin samples, a total of 400 strains of Aspergillus were isolated and analysed for OTA production. Twelve isolates (3%), from five raisin samples, produced OTA. These isolates were identified as Aspergillus carbonarius, based on morphological characteristics and multilocus sequence analysis. Levels of OTA produced by these isolates on raisin agar ranged from 0·9 to 15 μg g^?1. Conclusions: OTA is a common contaminant of raisin vineyards, but average levels are much lower than EU regulatory limits for dried fruit. The primary species responsible for OTA contamination in California raisins is A. carbonarius. Significance and Impact of the Study: This study illustrates that low‐level OTA contamination of raisins occurs in California and that ecological studies of A. carbonarius within the Aspergillus section Nigri population on raisins are warranted to monitor ochratoxigenic potential of the crop.     

Occurrence of aflatoxin B1 and ochratoxin A in Lebanese cultivated wheat

An extensive survey of filamentous fungi isolated from wheat grown and consumed in Lebanon and their capacity to produce aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) was conducted to assess fungi potential for producing these toxins in wheat. From the 468 samples of wheat kernel, collected at preharvest stage from different locations during 2008 and 2009 cultivation seasons, 3,260 fungi strains were isolated with 49.4% belonging to Penicillium spp. and 31.2% belonging to Aspergillus spp. Penicillium spp. was detected on wheat samples with a high amount of P. verrucosum (37.0%). Among the different Aspergillus spp. isolated, A. niger aggregate was predominant and constituted 37.3%. whereas the isolation rate of A. flavus and A. ochraceus was 32.2 and 25.6%, respectively. The ability to produce OTA and AFB₁ by isolates belonging to Aspergillus spp. and Penicillium spp. was analyzed by high performance liquid chromatography with fluorescence detector (HPLC-FLD). It was found that 57.0% of Penicillium spp. and 80% of A. ochraceus isolates tested produced OTA, respectively, at maximum concentrations of 53 and 65 μg/g CYA. As for the aflatoxinogenic ability, 45.3% of A. flavus produced AFB₁, with maximum concentration of 40 μg/g CYA. A total of 156 wheat samples were analyzed for the levels of OTA and AFB₁ by HPLC-FLD. The results showed that 23.7% were contaminated with OTA, at a concentration higher than 3 μg/kg and 35.2% of these samples were contaminated with AFB₁ at concentration higher than 2 μg/kg. The risks originating from toxin levels in wheat produced in Lebanon should be monitored to prevent their harmful effects on public health.     

Temperature and incubation time effects on growth and ochratoxin A production by Aspergillus sclerotioniger and Aspergillus lacticoffeatus on culture media

Aims: As there is no knowledge of the influence of abiotic factors on the two new ochratoxin A (OTA)‐producing species Aspergillus sclerotioniger and Aspergillus lacticoffeatus, the aim of this study was to evaluate the effect of temperature and incubation time on growth and OTA production by these species on culture media. Methods and Results: The study was carried out on yeast extract sucrose agar (YES) and Czapek yeast extract agar (CYA) incubated at ten different temperatures from 5 to 50°C (at 5°C intervals). Growth assessment and OTA production were determined after 5, 10, 15, 20 and 30 days of incubation at each temperature. Aspergillus sclerotioniger grew from 10 to 35°C; OTA was detected from 10 to 35°C and the highest concentration was achieved at 15°C in CYA. Aspergillus lacticoffeatus grew from 10 to 45°C; OTA was detected from 15 to 45°C, and the maximum concentration was produced after 5 days at 25°C in YES. Conclusions: The studied species can produce OTA over a wide range of temperatures and significant amounts can be produced in only 5 days. Significance and Impact of the Study: This is the first report on the influence of ecophysiological factors on these two ochratoxigenic species. The pattern of effects of temperature on growth and OTA production by A. sclerotioniger and A. lacticoffeatus was similar to those reported for the closely related species Aspergillus carbonarius and Aspergillus niger, respectively. The two new OTA‐producing species have both been isolated from coffee beans, and the closely related ochratoxigenic species of section Nigri, A. carbonarius and A. niger are important sources of OTA in this substrate.