Aflatoxins and ochratoxin A in stored barley grain in Spain and impact of PCR-based strategies to assess the occurrence of aflatoxigenic and ochratoxigenic Aspergillus spp

Contamination of barley by moulds and mycotoxins results in quality and nutritional losses and represents a significant hazard to the food chain. The presence of aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) and ochratoxin A (OTA) in stored barley in Spain has been studied. Species-specific PCR assays were used for detection of Aspergillus flavus, A. parasiticus, A. ochraceus, A. steynii, A. westerdijkiae, A. carbonarius and A. niger aggregate in mycotoxin-positive barley samples at different incubation times (0, 1 and 2 days). Classical enumeration techniques (CFU/g) in different culture media for evaluation of Aspergillus in sections Flavi, Circumdati and Nigri were also used. One hundred and five barley kernel samples were collected in Spanish grain stores from 2008 to 2010, and analyzed using a previously optimized method involving accelerated solvent extraction, cleanup by immunoaffinity column, liquid chromatographic separation, post-column derivatization with iodine and fluorescence detection. Twenty-nine samples were contaminated with at least one of the studied mycotoxins. AFB1, AFB2, AFG1, AFG2, and OTA were detected in 12.4%, 2.9%, 4.8%, 2.9%, and 20% of the samples, respectively. Aflatoxins and OTA co-occurred in 4.8% of the samples. Maximum mycotoxin levels (ng/g) were 0.61 (AFB1), 0.06 (AFB2), 0.26 (AFG1), 0.05 (AFG2), and 2.0 (OTA). The results of PCR assays indicated the presence of all the studied species, except A. westerdijkiae. The PCR assays showed high levels of natural contamination of barley with the studied species of Aspergillus which do not correspond to the expected number of CFU/g in the cultures. These results suggest that a high number of non-viable spores or hyphae may exist in the samples. This is the first study carried out on the levels of aflatoxins and OTA in barley grain in Spain. Likewise, this is the first report on the presence of aflatoxigenic and ochratoxigenic Aspergillus spp. in barley grain naturally contaminated with those mycotoxins using a species-specific PCR approach.     

Survey for aflatoxins, ochratoxin A, zearalenone and fumonisins in maize imported into the United Kingdom

This survey examined 140 samples of raw maize as received at ports or at major maize mills in the UK and 12 after initial cleaning. Samples were examined for aflatoxins B₁, B₂, G₁ and G₂, ochratoxin A, zearalenone and fumonisins B₁, B₂ and B₃ using fully validated analytical HPL C methods with detection limits of 0.1 mu g/kg for each aflatoxin and ochratoxin A, 4 mu g/ kg for zearalenone and 10 mu g/kg for each fumonisin. 95.0% and 92.1% of samples met the new EC statutory maximum permissible level for total aflatoxins and aflatoxin B₁ respectively. The maximum concentration of ochratoxin A found was 1.5 mu g/kg. Zearalenone and fumonisins were detected in almost every sample with 41.7% of maize containing more than 100 mu g/kg of zearalenone and 48% of samples containing more than 1000 mu g/kg total fumonisins. Initial cleaning of raw maize reduced aflatoxin concentrations by about 40% and total fumonisins by 32%.     

化学发光免疫分析方法检测粮食谷物中赭曲霉毒素A残留

目的建立粮食谷物中赭曲霉毒素A(ochratoxin A,OTA)残留检测的化学发光免疫分析方法。方法人工改造OTA半抗原、制备包被原和多克隆抗体工作液,优化检测步骤,验证各项评价指标,比较化学发光免疫分析方法和高效液相色谱法(high performance liquid chromatography,HPLC)的实际样本检测结果的一致性,以此来实现化学发光免疫分析方法的建立。结果 50%抑制浓度(IC_(50))为0.278μg/L;OTA的最低检出限为5.0μg/kg,样本添加回收率在65.4%到115.8%之间,批内、批间相对标准偏差15%。同时,通过对实际样本检测,证明化学发光免疫分析方法具有显著的准确度和可靠性。结论基于化学发光免疫分析方法能够实现粮食作物中的OTA残留的快速检测,灵敏度高、成本低、时间短,为食品中霉菌毒素残留的快速检测提供了新的技术参考。     

Co-occurrence of aflatoxins, ochratoxin A and zearalenone in barley from a northern region of Spain

One-hundred and twenty-three barley samples from a region of Spain (Navarra) were analysed in order to evaluate the possible co-occurrence of aflatoxins (AFB1, AFG1, AFB2 and AFG2), ochratoxin A (OTA) and zearalenone (ZEA). The results indicated that 80% of the samples presented detectable, although very low levels, of two or more mycotoxins. The most frequent combinations were AFB1 and OTA; AFB1, ZEA and OTA; and AFB1 and ZEA. In general, the statistical study did not show significant differences between levels or incidence for the mycotoxins in different years of harvest, variety of barley, farming or origin. The calculated values for daily intake were low and the risk to consumers could be assumed to be very low. However, the co-occurrence of several mycotoxins, and therefore synergic or additive effects, should be taken into account when determining permitted levels or risk assessment.     

Validation of a UHPLC-FLD method for the simultaneous quantification of aflatoxins,ochratoxin A and zearalenone in barley

A fast and simple UHPLC-FLD method has been developed for the simultaneous determination in barley of aflatoxins (B1, G1, B2 and G2), ochratoxin A (OTA) and zearalenone (ZEA), some of the most important mycotoxins due to their toxicity and occurrence. The procedure is based on the extraction of the six mycotoxins with a mixture of acetonitrile and water, and the purification of the extract with immunoaffinity columns before analysis. Detection of AFB1 and AFG1 is improved using a photochemical reaction. The method has been validated with satisfactory results. Limits of detection were 340 ng kg⁻¹ for ZEA, 13 ng kg⁻¹ for OTA and varied from 0.5 to 15 ng kg⁻¹ for aflatoxins. Recovery percentages were between 78.2% and 109.2%. After being validated, the method has been successfully applied to 20 barley samples cultivated in a region of northern Spain (Navarra).     

Trichothecenes, ochratoxin A and zearalenone contamination and Fusarium infection in Finnish cereal samples in 1998

The occurrences and concentrations of trichothecenes, ochratoxin A and zearalenone in Finnish cereal samples are presented in this study. Furthermore, infections by moulds, especially Fusarium contamination of grains in the same samples, are reported. In total 68 cereal samples, including 43 rye, 4 wheat, 15 barley and 6 oats samples, were collected after a cool and very rainy growing season in 1998. A gas chromatograph combined with a mass spectrometric detector was used for determination of seven different trichothecenes. A high performance liquid chromatograph with a fluorescence detector was used for ochratoxin A and zearalenone determination. For the identification of moulds, the grain samples were incubated and the moulds were isolated and identified by microscopy. The analytical methods were validated for mycotoxin analysis and they were found to be adequately reliable and sensitive. Heavy rainfalls in the summer and autumn of 1998 caused abundant Fusarium mould infection in Finnish cereals, particularly in rye. Fusarium avenaceum was the most common Fusarium species found in cereals. However, the mycotoxin concentrations were very low and only deoxynivalenol, nivalenol and HT-2 toxin were detected. Deoxynivalenol was detected in 54 samples in the concentration range 5-111 µg/kg. Nivalenol and HT-2 toxin were detected in three and two samples, respectively, in the concentration range 10-20 µg/kg.     

Deoxynivalenol and ochratoxin A in German wheat and changes of level in relation to storage parameters

The occurrence of the mycotoxins deoxynivalenol (DON) and ochratoxin A (OTA) in the winter wheat of 1997 and 1998 grown under organic farming conditions was investigated using ELISAs (R-Biopharm®) for quantification. The influence of delayed drying of the grain after harvest on the development of DON and OTA was determined in storage trials (moisture: 17% and 20%; temperature: 20°C; duration: four and six weeks). The Tox5 PCR assay was used both to detect Fusarium species with the potential to produce trichothecenes and as a measure of their relative DNA content during the storage trials. The intensity of the PCR signals was correlated with the DON concentration. Fusarium species were identified microscopically by standard methods. All the freshly harvested grain samples were contaminated with DON and showed further increases in the DON concentration during storage. OTA contamination was found in 14.3% of the 1997 samples and in 24.1% of the 1998 samples. OTA increased during storage trials of the 1997 samples but not in the 1998 samples.     

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Determination of ochratoxin A in brewing materials and beer by ultra performance liquid chromatography with fluorescence detection

In 2008–2009 the total set of 237 samples of malting barley, malt, hop, wort, and beer was analysed for ochratoxin A (OTA) contamination using the ultra performance liquid chromatography (UPLC) coupled to fluorescence detection (FLD). The UPLC method is a fast technique with low limits of detection and quantification (LOD and LOQ) compared to other methods used routinely. LOD and LOQ values were 0.0003 and 0.001 ng/ml for beer, 0.05 and 0.2 μg/kg for barley and malt, 0.16 and 0.5 μg/kg for hop, respectively.     

Determination of ochratoxin A in wheat after clean-up through a DNA aptamer-based solid phase extraction column

A DNA aptamer with high affinity and specificity to ochratoxin A (OTA) was conjugated to a coupling gel and used as sorbent for the preparation of solid phase extraction (SPE) columns. The SPE columns packed with 300 μl oligosorbent (24 nmol DNA) showed a linear (r = 0.999) behaviour in the range of 0.4–500 ng OTA. After optimisation of the extraction step, SPE columns were used for clean-up of OTA from wheat prior to liquid chromatographic (HPLC) analysis with fluorescence detection (FLD). Average recoveries from wheat samples spiked at levels of 0.5–50 ng/g ranged from 74% to 88% (relative standard deviation <6%) with limits of detection and of quantification of 23 and 77 pg/g, respectively. The comparative HPLC/FLD analyses of 33 naturally contaminated durum wheat samples cleaned-up on both aptamer-SPE and immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance.     

Survey of Argentinean human plasma for ochratoxin A

The presence of ochratoxin A (OTA) in human blood has been reported for many countries, especially in Europe. However, so far no report exists concerning such a presence in Argentina. The aim of this study was to assess OTA concentration in human plasma in two different areas of Buenos Aires province. OTA was determined by high-performance liquid chromatography (HPLC) in 199 plasma samples from blood donors in Mar del Plata and 236 from General Rodríguez. Solid-phase extraction with Bakerbond® C-18 cartridge and a final purification with Ochraprep® immunoaffinity columns was employed. The limit of quantification of ochratoxin A was 0.019ngml^?1 and the confirmation of OTA was by formation of ochratoxin A methyl ester. The results showed that 63.8% of human plasma samples from Mar del Plata and 62.3% from General Rodríguez were positive for OTA, with Winsorized means of 0.15 and 0.43ngml^?1, respectively. It is important to continue the research to detect the foods responsible of the presence of OTA in plasma.     

Survey of Canadian retail coffees for ochratoxin A

One-hundred and one specimens of coffee were gathered from retail outlets across Canada and analysed for ochratoxin A. Seventy-one specimens were roasted beans or roasted ground coffee, and 30 were instant (or 'soluble') coffees. All samples were extracted with methanol-sodium bicarbonate. The extracts were cleaned up either by immunoaffinity column chromatography or by a combination of solid-phase extraction and immunoaffinity column chromatography. Ochratoxin A was quantified by liquid chromatography (LC) with fluorescence detection. The minimum quantifiable level was 0.1 ng g^-1. Ochratoxin A was present, above the minimum quantifiable level, in 42 (59%) of 71 beans and ground coffee and in 20 (67%) of 30 instant coffees. The mean ochratoxin A level in the positive samples of beans and ground coffee was 0.6 ng g^-1, and the mean level in the positive samples of instant coffee was 1.1 ng g^-1.     

Simple liquid chromatography assay for analyzing ochratoxin A in bovine milk

Ochratoxin A (OTA) is a mycotoxin with teratogenic and carcinogenic properties. Animal intake of feedstuffs contaminated with OTA may cause that some residues may be found in bovine milk, therefore, its analysis requires a highly sensitive, simple and precise technique.     

Occurrence of ochratoxin A in Danish wheat and rye, 1992-99

Ochratoxin A concentrations in rye and wheat in Denmark for 1992-99 are reported. The results show that the concentration of ochratoxin A is higher in rye than in wheat for both conventionally and organically grown rye and wheat. The levels in organically grown rye are higher than in conventionally grown based on multiyear mean contents. However, the difference between the two groups of cereals has decreased since the Danish food-monitoring system for ochratoxin A was started in 1986; 2.0% of all samples exceeded the Danish maximum limit of 5 µg kg -1 introduced in 1995. For rye samples, 3.2% exceeded the maximum limit, and for wheat samples, 0.5% exceeded the maximum limit.     

Potential ochratoxin A producers from wine grapes in Argentina and Brazil

The aim was to identify the normal mycoflora in wine grapes from Argentina and Brazil. We collected 50 grapes samples from Malbec and Chardonnay varieties in each country during the 1997-98 harvest. Yeasts were a major component of the fungal population, and the most frequent genera of filamentous fungi isolated were: Aspergillus, Penicillium and Botrytis. Other genera identified (in decreasing order) were: Phythophthora, Moniliella, Alternaria and Cladosporium. From grapes, the mean frequency of filamentous fungi ranged from 1.3 × 10⁴ to 5.4 × 10⁶ CFUg^-1. We isolated 48 Aspergillus niger strains from Argentinian grape, of which eight could produce ochratoxin A. Sixteen of 53 A. niger strains from Brazilian grapes produced ochratoxin A. The results indicate that similar mycobiota were isolated from Argentinian and Brazilian wine grapes and there could be ochratoxin A production in this substrate.     

葡萄中碳黑曲霉的分离及其产生赭曲霉毒素A的研究

碳黑曲霉(Aspergillus carbonarius)是葡萄中产生赭曲霉毒素A(Ochratoxin A,OTA)的重要菌株.采集烟台赤霞珠(Cabernet sauvignon)葡萄,接种于孟加拉红培养基,从中分离到7株黑曲霉群(Aspergillus section black group)真菌,其中3株鉴定属于碳黑曲霉种,占黑色曲霉的43.8%.此3菌株分别接种在粮粒培养基上,静置培养,全部产生OTA,最高浓度达到1300μg/kg,而且,其中2株碳黑曲霉菌株在可可浆培养基上产生荧光,而从葡萄样品中未检出OTA.     

Effects of ochratoxin A on micronucleus frequenncy in human lymphocytes

Ochratoxin A (OTA( a nephrotoxic and carcinogenic mycotoxin, was investigated to examine its potency to induce micronuclei (MN) in cultured human lymphocytes. Lymphocyte cultures were treated for the last 48 h with OTA at concentrations of 25 μM , 10 μM , 1 μM , 100 nM , 10 nM , 1 nM , and 100 pM and absolute ethanol. At the highest concentration, OTA was found to induce MN in cytokinesis‐blocked lymphocytes (p < 05). The 25 μM OTA concentration also led to a clear decrease in the percentage of binucleated cells, probably due to cytotoxicity. OTA at the other concentrations tested did not induce MN frequency. These results indicate that a high concentration of OTA is genotoxic in cultured human lymphocytes.     

Factors affecting the presence of ochratoxin A in wines

Ochratoxin A (OTA) are synthesized mainly by different species of Aspergillus and Penicillium being its human toxicological effects reflected in different countries due to the consumption of different foods and beverages such as red, white, rose, and special wines. This review presents an overview of the direct (meteorological conditions, grape cultivation, and wine-making techniques) and indirect (latitude, year of production, use of pesticides, presence of spoilage microorganisms, conditions of storage of the harvested grapes, type of maceration, and conditions of fermentation), factors affecting the presence of OTA in wines.     

Environmental factors affect efficacy of some essential oils and resveratrol to control growth and ochratoxin A production by Penicillium verrucosum and Aspergillus westerdijkiae on wheat grain

This study determined the efficacy of three essential oils (bay, clove and cinnamon oil) and the antioxidant resveratrol (0–500 μg g⁻¹) on the control of growth and ochratoxin A (OTA) production by Penicillium verrucosum and Aspergillus westerdijkiae (=A. ochraceus) under different water activity (a_w, 0.90, 0.95, 0.995), and temperature (15, 25 °C) conditions on irradiated wheat grain. The most effective treatment (resveratrol) was then tested on natural grain. The ED₅₀ values for growth inhibition by the oils were 200–300 μg g⁻¹ at the a_w and the temperatures tested. For resveratrol, this varied from <50 μg g⁻¹ at 0.90–0.95 a_w to >350 at 0.995a_w at both temperatures. The ED₅₀ values for the control of OTA were slightly lower than for control of growth, with approx. 200 μg g⁻¹ required for the oils and 50–100 μg g⁻¹ of the antioxidant, at 0.90/0.95a_w and both temperatures. In wet grain (0.995a_w), higher concentrations were required. For growth there were statistically significant effects of single-, two- and three-way interactions between treatments except for concentration×temperature and concentration×temperature×essential oil/antioxidant treatment. For OTA control, statistically significant treatments were a_w, temperature×a_w, concentration×temperature, treatment×concentration, and three-way interaction of concentration×a_w×treatment for P. verrucosum and A. westerdijkiae. Subsequent studies were done with the best treatment (resveratrol, 200 μg g⁻¹) on natural wheat grain with either P. verrucosum or A. westerdijkiae at 0.85–0.995a_w and 15/25 °C over 28 days storage. This showed that the populations of the mycotoxigenic species and OTA contamination could be reduced by >60% by this treatment at the end of the storage period.