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Concept: Ochratoxin A

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Ochratoxin A (OTA) is a mycotoxin found in a wide range of food and feedstuffs. Intake of OTA-contaminated food causes health concern due to the harmful effects reported on humans and animals. Much effort is currently devoted to set up and optimise highly sensitive and accurate methods of OTA analysis. This work describes the comparison of fluorescence-based immunosensing strategies for the analysis of OTA. First, an indirect competitive fluoroimmunoassay was designed and optimised. The assay enabled the quantification of the toxin at the levels set by the European legislation. Then, a flow-immunoassay based on kinetic exclusion measurements was developed. It showed the theoretical lowest limit of detection enabled by the affinity of the anti-OTA antibody (IC(80)=12ngL(-1) in the assay solution). Wine and cereal samples were analysed using the optimised flow system. No significant matrix effects were observed after simple pre-treatment of wine and OTA extraction from corn-flakes samples. This simple and highly sensitive automated biosensing-system allows OTA quantification in food and beverages. It is envisaged as a powerful tool for rapid and reliable toxin screening.

Concepts: Scientific method, Energy, Measurement, Mycotoxin, Cereal, Search engine optimization, Ochratoxin, Ochratoxin A

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A new sample preparation procedure, termed pH-controlled dispersive liquid-liquid microextraction (pH-DLLME), has been developed for the analysis of ionisable compounds in highly complex matrices. This DLLME mode, intended to improve the selectivity and to expand the application range of DLLME, is based on two successive DLLMEs conducted at opposite pH values. pH-DLLME was applied to determination of ochratoxin A (OTA) in cereals. The hydrophobic matrix interferences in the raw methanol extract (disperser, 1mL) were removed by a first DLLME (I DLLME) performed at pH 8 to reduce the solubility of OTA in the extractant (CCl(4), 400μL). The pH of the aqueous phase was then adjusted to 2, and the analyte was extracted and concentrated by a second DLLME (extractant, 150μL C(2)H(4)Br(2)). The main factors influencing the efficiency of pH-DLLME including type and volume of I DLLME extractant, as well as the parameters affecting the OTA extraction by II DLLME, were studied in detail. Under optimum conditions, the method has detection and quantification limits of 0.019 and 0.062μgkg(-1), respectively, with OTA recoveries in the range of 81.2-90.1% (n=3). The accuracy of the analytical procedure, evaluated with a reference material (cereal naturally contaminated with OTA), is acceptable (accuracy of 85.6%±1.7, n=5). The applicability of pH-DLLME to the selective extraction of other ionisable compounds, such as acidic and basic pharmaceutical products was also demonstrated. The additional advantages of pH-DLLME are a higher selectivity and the extension of this microextraction technique to highly complex matrices.

Concepts: Acid, PH, Cereal, Extraction, PH indicator, Matrix, Ochratoxin, Ochratoxin A

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The occurrence of P. verrucosum and ochratoxin A (OTA) were surveyed for 3 and 4 years, respectively. A total of 250 samples was collected from an average of 30 farms during the 2011, 2012, 2013 and 2014 winter seasons. Most storage bins surveyed were typically 11 m high round bins made of corrugated, galvanized steel, with flat-bottoms and conical roofs. Samples of clumped grain contained the most P. verrucosum (p<0.05, n = 10) followed by samples taken from the first load (n = 24, mean = 147±87 CFU/g) and last load (n = 17, mean = 101±77 CFU/g). Five grain samples (2.2%) tested positive for OTA, citrinin and OTB at concentrations of 14.7±7.9, 4.9±1.9 and 1.2±0.7 ng/g, with only three samples exceeding 5 ng/g. Grain samples positive for OTA were related to moisture resulting from either condensation or migrating moist warm air in the bin or areas where precipitation including snow entered the bin. Bins containing grain and clumps contaminated with OTA were studied in detail. A number of statistically-significant risk factors for OTA contamination were identified. These included 1) grain clumps accumulated around or directly under manhole openings, 2) debris and residue of old grain or grain clumps collected from the bin walls or left on storage floor and augers and 3) grain clumps accumulated around side doors. Even when grain enters storage below the 14.5% threshold of moisture, condensation and moisture migration occurs in hotspots in modern corrugated steel storage bins. Hot spots of OTA contamination were most often in areas affected by moisture migration due to inadequate aeration and exposure to moisture from precipitation or condensation. Further, we found that the nature of the condensation affects the nature and distribution of small and isolated areas with high incidence of toxin contamination and/or P. verrucosum prevalence in the grain bins examined.

Concepts: Wheat, HotSpot, Penicillium, Ochratoxin, Great Lakes, Ochratoxin A, Hot-dip galvanizing, Corrugated galvanised iron

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Ochratoxin A (OTA) is a mycotoxin identified as a contaminant in grains and wine throughout the world, and convenient, rapid and sensitive detection methods for OTA have been a long-felt need for food safety monitoring. Herein, we presented a new competitive format based lateral flow strip fluorescent aptasensor for one-step determination of OTA in corn samples. Briefly, biotin-cDNA was immobilized on the surface of a nitrocellulose filter on the test line. Without OTA, Cy5-labeled aptamer combined with complementary strands formed a stable double helix. In the presence of OTA, however, the Cy5-aptamer/OTA complexes were generated, and therefore less free aptamer was captured in the test zone, leading to an obvious decrease in fluorescent signals on the test line. The test strip showed an excellent linear relationship in the range from 1 ng·mL-1 to 1000 ng·mL-1 with the LOD of 0.40 ng·mL-1, IC15 value of 3.46 ng·mL-1 and recoveries from 96.4% to 104.67% in spiked corn samples. Thus, the strip sensor developed in this study is an acceptable alternative for rapid detection of the OTA level in grain samples.

Concepts: Food, Mycotoxin, Cereal, Helix, Ochratoxin, Mycotoxins, Whole grain, Ochratoxin A

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This study was conducted to investigate the natural co-occurrence of 295 fungal and bacterial metabolites in 28 samples of dried date palm fruits collected from different shops distributed in Assiut Governorate, Upper Egypt in 2016. Extraction and quantification of the target analytes were done using the “dilute and shoot” approach followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. In total, 30 toxic fungal metabolites were detected. Among these metabolites, 4 types of ochratoxins including ochratoxin type A and B were quantified in 3 samples (11%) with a contamination range from 1.48 to 6070 μg/kg for ochratoxin A and from 0.28 to 692 μg/kg for ochratoxin B. In addition, fumonisin B2 was observed in 2 (7%) samples with contamination levels ranging from 4.99 to 16.2 μg/kg. The simultaneous detection of fumonisin B2 in the same contaminated samples with ochratoxins indicates the fungal attack by Aspergillus niger species during storage. Only 1 sample was contaminated with aflatoxin B1 (14.4 μg/kg) and B2 (2.44 μg/kg). The highest maximum concentration (90400 μg/kg) was for kojic acid that contaminated 43% of the samples. To the best of the authors' knowledge, this is the first report of the natural co-occurrence of fumonisin B2 and ochratoxin A and B in addition to a wide range of other fungal metabolites in date palm fruits.

Concepts: Fungus, Aspergillus flavus, Aflatoxin, Mycotoxin, Aspergillus, Ochratoxin, Mycotoxins, Ochratoxin A

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The environmental conditions reached during the ripening of dry-cured meat products favour the proliferation of moulds on their surface. Some of these moulds are hazardous to consumers because of their ability to produce ochratoxin A (OTA). Biocontrol using Debaryomyces hansenii could be a suitable strategy to prevent the growth of ochratoxigenic moulds and OTA accumulation in dry-cured meat products. The aim of this work was to evaluate the ability of two strains of D. hansenii to control the growth and OTA production of Penicillium verrucosum in a meat model under water activities (aw) values commonly reached during the dry-cured meat product ripening. The presence of D. hansenii strains triggered a lengthening of the lag phase and a decrease of the growth rate of P. verrucosum in meat-based media at 0.97 and 0.92 aw. Both D. hansenii strains significantly reduced OTA production (between 85.16 and 92.63%) by P. verrucosum in the meat-based medium at 0.92 aw. Neither absorption nor detoxification of OTA by D. hansenii strains seems to be involved. However, a repression of the expression of the non-ribosomal peptide synthetase (otanpsPN) gene linked to the OTA biosynthetic pathway was observed in the presence of D. hansenii. To confirm the protective role of D. hansenii strains, they were inoculated together with P. verrucosum Pv45 in dry-fermented sausage and dry-cured ham slices. Although P. verrucosum Pv45 counts were not affected by the presence of D. hansenii in both meat matrices, a reduction of OTA amount was observed. Therefore, the effect of D. hansenii strains on OTA accumulation should be attributed to a reduction at transcriptional level. Consequently, native D. hansenii can be useful as biocontrol agent in dry-cured meat products for preventing the hazard associated with the presence of OTA.

Concepts: Protein, Enzyme, Yeast, Mycotoxin, Penicillium, Ochratoxin, Ochratoxin A, Meat by-product

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Ochratoxin A (OTA) is a potent nephrotoxin and carcinogen which is found in a wide variety of common foods and beverages and it is produced by several species of Aspergillus and Penicillium. Ochratoxin α (OTα), a major metabolite of OTA, has also been reported to occur in cultures of OTA-producing species. However there is some controversial about the participation of OTα in the biosynthesis of OTA, mainly because its biosynthesis pathway has not yet been completely characterized. Aspergillus carbonarius is the main responsible source of ochratoxin A (OTA) in food commodities such as wine, grapes or dried vine fruits from main viticultural regions worldwide. However, little is known about the presence of OTα in isolates of A. carbonarius. In this study we evaluated the effects of temperature and incubation time on OTα production by both OTA and non-OTA-producing strains of A. carbonarius. OTA and OTα were detected on the basis of HPLC fluorometric response compared with that of their standards and confirmed by HPLC-MS in selected samples. The non-OTA-producing strains did produce neither OTA nor OTα at any of the conditions tested. The OTA-producing strains studied were able to produce both OTA and OTα in most of the conditions tested. In general, higher amounts of OTA than OTα were produced, but a positive correlation in the production of these two metabolites was detected. The lack of production of both OTA and OTα in the non-OTA-producing strains could be caused by the presence of silent genes or by mutations in functional or regulatory genes involved in OTA production.

Concepts: HIV, DNA, Metabolism, Mycotoxin, Cultural studies, Grape, Ochratoxin, Ochratoxin A

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Here we report a surprising discovery that Co/Fe-MOFs possess intrinsic electrocatalytic activities for thionine. The catalytic abilities of MOFs depend strongly on their μ3-O linked trigonal prism structures. Using NH2-Co-MOF as an example, the high-performance of electrocatalysis towards thionine for ultrasensitive electrochemical detection of ochratoxin A is first established.

Concepts: Electrochemistry, Catalysis, Catalytic converter, Chemical equilibrium, Activity, Ochratoxin, Ochratoxin A

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Ochratoxin A (OTA) is one of the most widespread and dangerous food contaminants. Therefore, rapid, label-free and precise detection of low OTA concentrations requires novel sensing elements with advanced bio-analytical properties. In the present paper we report photoluminescence (PL) based immunosensor for the detection of OTA. During the development of immunosensor photoluminescent ZnO nanorods (ZnO-NRs) were deposited on glass substrate. Then the ZnO-NRs were silanized and covalently modified by Protein-A (Glass/ZnO-NRs/Protein-A). The latest structure was modified by antibodies against OTA (Anti-OTA) in order to form OTA-selective layer (Glass/ZnO-NRs/Protein-A/Anti-OTA). In order to improve immunosensors selectivity the surface of Glass/ZnO-NRs/Protein-A/Anti-OTA was additionally blocked by BSA. Formed Glass/ZnO-NRs/Protein-A/BSA&Anti-OTA structures were integrated within portable fiber optic detection system, what is important for the development of low cost and portable immunosensors. The immunosensor has been tested in a wide range of OTA concentrations from 10(-4)ng/ml until 20ng/ml. Interaction isotherms were derived from analytical signals of immunosensor. Association constant and Gibbs free energy for the interaction of Glass/ZnO-NRs/Protein-A/Anti-OTA with OTA were calculated, analyzed and compared with some other related results. Sensitivity range and limit of detection were determined as 0.1-1ng/ml and 10(-2)ng/ml, respectively. Interaction kinetics of ZnO-NRs with OTA was evaluated. Response time of the immunosensor toward OTA was in the range of 500-800s. Some insights related to the mechanism of PL-signal generation are proposed and discussed.

Concepts: Energy, Chemical equilibrium, Thermodynamics, Entropy, Gibbs free energy, Kinetics, Ochratoxin A, Photoluminescence

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Regarding teratogenic, carcinogenic, and immunotoxic nature of ochratoxin A (OTA), selective and sensitive monitoring of this molecule in food samples is of great importance. In recent years, various methods have been introduced for detection of OTA. However, they are usually time-consuming, labor-intensive and expensive. Therefore, these parameters limited their usage. The emerging method of detection, aptasensor, has attracted more attention for OTA detection, due to distinctive advantages including high sensitivity, selectivity and simplicity. In this review, the new developed aptasensors for detection of OTA have been investigated. We also highlighted advantages and disadvantages of different types of OTA aptasensors. This review also takes into consideration the goal to find out which designs are the most rational ones for highly sensitive detection of OTA.

Concepts: DNA, Sensitivity and specificity, Economics, Cost, Ochratoxin, Ochratoxin A