Ochratoxin A In Food Research Articles

Shark anti-idiotypic variable new antigen receptor specific for an alpaca nanobody: Exploration of a nontoxic substitute to ochratoxin A in immunoassay

Nontoxic substitutes to mycotoxins can facilitate the development of eco-friendly immunoassays. To explore a novel nontoxic substitute to ochratoxin A (OTA), this study screened shark anti-idiotypic variable new antigen receptors (VNARs) against the alpaca anti-OTA nanobody Nb28 through phage display. After four rounds of biopanning of a naïve VNAR phage display library derived from six adult Chiloscyllium plagiosum sharks, one positive clone, namely, P-3, was validated through a phage enzyme–linked immunosorbent assay (phage ELISA). The recombinant anti-idiotypic VNAR AId-V3 was obtained by prokaryotic expression, and the interactions between Nb28 and AId-V3 were investigated via computer-assisted simulation. The affinity of AId-V3 for Nb28 and its heptamer Nb28-C4bpα was measured using Biacore assay. Combining Nb28-C4bpα with AId-V3, a novel direct competitive ELISA (dcELISA) was developed for OTA analysis, with a limit of detection of 0.44 ng/mL and a linear range of 1.77–32.25 ng/mL. The good selectivity, reliability, and precision of dcELISA were confirmed via cross-reaction analysis and recovery experiments. Seven commercial pepper powder samples were tested using dcELISA and validated using high-performance liquid chromatography. Overall, the shark anti-idiotypic VNAR was demonstrated as a promising nontoxic substitute to OTA, and the proposed method was confirmed as a reliable tool for detecting OTA in food.

Exonuclease III assisted electrochemical aptasensor simultaneous detection of aflatoxin B1 and ochratoxin a in grains

The synergistic effect of multiple mycotoxins in cereals increases their toxicity. Therefore, the simultaneous detection of multiple mycotoxins in cereals is of great importance. Exonuclease III (Exo III) assisted electrochemical aptasensor has been used for mycotoxin detection, but simultaneous detection of two mycotoxins has not been previously reported. By utilizing Exo III assisted strand displacement, this study developed an electrochemical aptasensor strategy for the simultaneous detection of aflatoxin B1 (AFB1) and ochratoxin A (OTA). This method demonstrates excellent detection of AFB1 and OTA within the range of 0.001–500 ng mL−1, with detection limits as low as 0.229 pg mL−1 for AFB1 and 0.564 pg mL−1 for OTA. The electrochemical aptasensor was successfully applied to analyze grain samples, and the sensor has good specificity, stability and repeatability. The adapter provides simultaneous detection of AFB1 and OTA in food in a fast, simple and highly sensitive platform.

Ready-to-use ratiometric bioluminescence immunosensor for detection of ochratoxin a in pepper

Rapid, portable, and accurate detection tools for monitoring ochratoxin A (OTA) in food are essential for the guarantee of food safety and human health. Herein, as a proof-of-concept, this study proposed a ratiometric bioluminescence immunosensor (RBL-immunosensor) for homogeneous detection of OTA in pepper. The construct of the RBL-immunosensor consists of three components, including the large fragment of the split nanoluciferase (NanoLuc)-tagged nanobody (NLg), the small fragment of the split NanoLuc-tagged mimotope peptide heptamer (MPSm), and the calibrator luciferase (GeNL). The specific nanobody-mimotope peptide interaction between NLg and MPSm induces the reconstitution of the NanoLuc, which catalyzes the Nano-Glo substrate and produces a blue emission peak at 458 nm. Meanwhile, GeNL can produce a green emission peak at 518 nm upon substrate conversion via bioluminescent resonance energy transfer (BRET). Therefore, the concentration of OTA can be linked to the variation of the bioluminescence signal (λ458/λ518) measured by microplate reader and the variation of the blue/green ratio measured by smartphone via the competitive immunoreaction where OTA competes with MPSm to bind NLg. The immunosensor is ready-to-use and works by simply mixing the components in a one-step incubation of 10 min for readout. It has a limit of detection (LOD) of 0.98 ng/mL by a microplate reader and an LOD of 1.89 ng/mL by a smartphone. Good selectivity and accuracy were confirmed for the immunosensor by cross-reaction analysis and recovery experiments. The contents of OTA in 10 commercial pepper powder samples were tested by the RBL-immunosensor and validated by high-performance liquid chromatography. Hence, the ready-to-use RBL-immunosensor was demonstrated as a highly reliable tool for detection of OTA in food.

Plasmonic enzyme immunoassay via nanobody-driven controllable aggregation of gold nanoparticles for detection of ochratoxin A in pepper

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H2O2), and then HRP catalyzes H2O2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food.

Comprehensive Insights into Ochratoxin A: Occurrence, Analysis, and Control Strategies.

Ochratoxin A (OTA) is a toxic mycotoxin produced by some mold species from genera Penicillium and Aspergillus. OTA has been detected in cereals, cereal-derived products, dried fruits, wine, grape juice, beer, tea, coffee, cocoa, nuts, spices, licorice, processed meat, cheese, and other foods. OTA can induce a wide range of health effects attributable to its toxicological properties, including teratogenicity, immunotoxicity, carcinogenicity, genotoxicity, neurotoxicity, and hepatotoxicity. OTA is not only toxic to humans but also harmful to livestock like cows, goats, and poultry. This is why the European Union and various countries regulate the maximum permitted levels of OTA in foods. This review intends to summarize all the main aspects concerning OTA, starting from the chemical structure and fungi that produce it, its presence in food, its toxicity, and methods of analysis, as well as control strategies, including both fungal development and methods of inactivation of the molecule. Finally, the review provides some ideas for future approaches aimed at reducing the OTA levels in foods.

Practical Strategies to Reduce Ochratoxin A in Foods.

Ochratoxin A (OTA), a potent nephrotoxin, is one of the most deleterious mycotoxins, with its prevalence in agricultural crops and their processed foods around the world. OTA is a major concern to food safety, as OTA exposure through dietary intake may lead to a significant level of accumulation in the body as a result of its long half-life (about 35 days). Its potent renal toxicity and high risk of exposure as well as the difficulty in controlling environmental factors OTA production has prompted the need for timely information on practical strategies for the food industry to effectively manage OTA contamination during food processing. The effects of various food processes, including both nonthermal and thermal methods, on the reduction in OTA were summarized in this review, with emphasis on the toxicity of residual OTA as well as its known and unknown degradation products. Since complete removal of OTA from foodstuffs is not feasible, additional strategies that may facilitate the reduction in OTA in food, such as adding baking soda and sugars, was also discussed, so that the industry may understand and apply practical measures to ensure the safety of its products destined for human consumption.

Food Safety Risk: Ochratoxin A in Türkiye

Ochratoxin A (OTA) is a mycotoxin produced by fungi of the genus Aspergillus and Penicillium. OTA causes damage to the kidneys and liver in experimental animals and is even classified as a possible human carcinogen. As an occasional phenomenon, many foods in a particular region may be contaminated with OTA. Regardless of whether it is a developed or developing country, OTA contamination poses both public health and economic risks. We have compiled studies on OTA in food and humans (milk and blood) in Turkey over the last 25 years and discussed what needs to be done to reduce the public health risk.

Combination of nanobody and peptidomimetic to develop novel immunoassay platforms for detecting ochratoxin A in cereals.

Mimotope-based immunoassays for mycotoxins eliminate the requirement for large amounts of mycotoxin standards for the chemosynthesis of artificial antigens. Herein, the nanobody-based magnetic beads were used to screen the mimotope (peptidomimetic) of ochratoxin A (OTA) from the phage-displayed peptide library. The interactions between nanobody and the most sensitive Y4 peptidomimetic were investigated by computer-assisted simulation and compared with those between nanobody and OTA. By combining the nanobody, the phage-displayed Y4 and alkaline phosphatase-tagged Y4 fusion protein as the competing antigens, were used to develop two novel immunoassay platforms (PN-ELISA and APN-ELISA). The two methods are advantageous in the use of nontoxic substitutes of OTA and avoiding the use of monoclonal antibodies. Moreover, good analytical performances of both methods were obtained and confirmed by liquid chromatography tandem mass spectrometry. Therefore, the proposed novel methods based on nanobody and peptidomimetic were demonstrated to be highly reliable for detecting OTA in food.

An accurate and ultrasensitive ratiometric electrochemical aptasensor for determination of Ochratoxin A based on catalytic hairpin assembly

Ochratoxin A (OTA) pollution in agricultural products has raised the pressing to develop sensitive, accurate and convenient detection methods. Herein, an accurate and ultrasensitive ratiometric electrochemical aptasensor was proposed based on catalytic hairpin assembly (CHA) for OTA detection. In this strategy, the target recognition and CHA reaction were both accomplished in the same system, which avoided tedious multi-steps operation and extra reagents, providing the advantage of convenience with only a one-step reaction and without enzyme. The labeled Fc and MB were used as the signal-switching molecules, avoiding various interferences and greatly improving the reproducibility (RSD: 3.197%). This aptasensor achieved trace-level detection for OTA with LOD of 81 fg/mL in the linear range of lower concentration (100 fg/mL-50 ng/mL). Moreover, this strategy was successfully applied to OTA detection in cereals with comparable results of HPLC-MS. This aptasensor provided a viable platform for accurate, ultrasensitive, and one-step detection of OTA in food.

Fluorescent Sensor Based on Magnetic Separation and Strand Displacement Amplification for the Sensitive Detection of Ochratoxin A.

Ochratoxin A (OTA) is a common mycotoxin, and it is a significant threat to human health throughout the food chain. In this study, a sensitive and specific fluorescent sensor based on magnetic separation technology combined with chain displacement amplification was developed for fast and easy detection of OTA in food. The designed strand displacement amplification can improve the sensitivity for the detection, and the magnetic nanomaterials can provide a large surface area, thus enhancing the capture efficiency of the target from the sample. Based on those designs, the experimental results showed that the proposed method displayed excellent performance. The linearity range was 0.5-128.0 ng/mL. The detection limit was 0.125 ng/mL; the relative standard deviations were 3.92-7.71%. Additionally, the developed method was satisfactorily applied to determine OTA in wheat, corn, and red wine samples at three spiked levels (1.0, 8.0, and 64.0 ng/mL). The recoveries ranged from 85.45 to 107.8% for wheat flour, 101.34 to 108.35% for corn flour, and 91.15 to 93.80% for red wine, respectively. Compared with high-performance liquid chromatography, the proposed method showed a lower limit of detection and equal recovery. Hence, the designed method is a potential and good detecting tool for OTA residue analysis in complex matrix samples.

Magnetic covalent organic framework for effective solid-phase extraction and HPLC determination of ochratoxin A in food

Ochratoxin A (OTA) exposure in food poses great threat to human health. Magnetic solid phase extraction (MSPE) is an effective sample pre-treatment method based on liquid chromatographic analysis to monitor OTA in food. Herein, magnetic covalent organic frameworks (Fe3O4@COFs) nanospheres were prepared and well characterized. The efficient and selective extraction of OTA relied on large specific surface and inherent porosity of COFs and the hydrogen bonding, π-π and hydrophobic interactions between COF shell and OTA. The MSPE parameters were investigated in detail. Under the optimal MSPE conditions, the developed Fe3O4@DhaTab-based-MSPE-HPLC-FLD method for OTA displayed good linearity (0.1-800 μg L−1) with R2 (0.996) and low limit of detection (0.03 μg L−1). The proposed method was applied to determine OTA in beer, Chinese Baijiu and vinegar with satisfactory recoveries (72–111%) and relative standard deviations (0.2 to 5.5%).

Rapid Identification and Analysis of Ochratoxin-A in Food and Agricultural Soil Samples Using a Novel Semi-Automated In-Syringe Based Fast Mycotoxin Extraction (FaMEx) Technique Coupled with UHPLC-MS/MS.

In this work, a fast mycotoxin extraction (FaMEx) technique was developed for the rapid identification and quantification of carcinogenic ochratoxin-A (OTA) in food (coffee and tea) and agricultural soil samples using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) detection. The FaMEx technique advancement is based on two plastic syringes integrated setup for rapid extraction and its subsequent controlled clean-up process. In the extraction process, a 0.25-g sample and extraction solvent were added to the first syringe barrel for the vortex-based extraction. Then, the extraction syringe was connected to a clean-up syringe (pre-packed with C18, activated carbon, and MgSO4) with a syringe filter. Afterward, the whole set-up was placed in an automated programmable mechanical set-up for controlled elution. To enhance FaMEx technology performance, the various influencing sample pretreatment parameters were optimized. Furthermore, the developed FaMEx method indicated excellent linearity (0.9998 and 0.9996 for coffee/tea and soil) with highly sensitive detection (0.30 and 0.29 ng/mL for coffee/tea and soil) and quantification limits (1.0 and 0.96 for coffee/tea and soil), which is lower than the toxicity limit compliant with the European Union regulation for OTA (5 ng/g). The method showed acceptable relative recovery (84.48 to 100.59%) with <7.34% of relative standard deviation for evaluated real samples, and the matrix effects were calculated as <-13.77% for coffee/tea and -9.7 for soil samples. The obtained results revealed that the developed semi-automated FaMEx/UHPLC-MS/MS technique is easy, fast, low-cost, sensitive, and precise for mycotoxin detection in food and environmental samples.

Unveiling ochratoxin a controlling and biodetoxification molecular mechanisms: Opportunities to secure foodstuffs from OTA contamination.

Anarchic growth of ochratoxin A (OTA) producing fungi during crop production, prolonged storage, and processing results in OTA contamination in foodstuffs. OTA in food exacerbates the risk of health and economic problems for consumers and farmers worldwide. Although the toxic effects of OTA on human health have not been well established, comprehensive preventive and remedial measures will be essential to eliminate OTA from foodstuffs. Strict regulations, controlling OTA at pre- or post-harvest stage, and decontamination of OTA have been adopted to prevent human and animal OTA exposure. Biological control of OTA and bio-decontamination are the most promising strategies due to their safety, specificity and nutritional value. This review addresses the current understanding of OTA biodegradation mechanisms and recent developments in OTA control and bio-decontamination strategies. Additionally, this review analyses the strength and weaknesses of different OTA control methods and the contemporary approaches to enhance the efficiency of biocontrol agents. Overall, this review will support the implementation of new strategies to effectively control OTA in food sectors. Further studies on efficacy-related issues, production issues and cost-effectiveness of OTA biocontrol are to be carried out to improve the knowledge, develop improved delivery technologies and safeguard the durability of OTA biocontrol approaches.

Reduction of ochratoxin A from contaminated food by Lactobacillus rhamnosus Bm01

Ochratoxin A (OTA) is a secondary fungal metabolite produced by Aspergillus and Penicillium spp. OTA contamination of food and animal feed has been attracting increasing concern in view of its toxicity, such as nephrotoxic, teratogenic, embryotoxic, genotoxic, neurotoxic, carcinogenic, and immunosuppressive effects. The widespread contamination of OTA in food and multiple toxicity of OTA have promoted research on detoxifying OTA in food or feed. The use of lactic acid bacteria (LAB) to remove OTA in food is a highly innovative and promising method compared with traditional methods. In this research, we screened an LAB strain, namely, Lactobacillus rhamnosus Bm01, which can remove 83.58% of OTA (50 ng/mL) within 48 h. The physiological mechanism of OTA removal by L. rhamnosus Bm01 was investigated. L. rhamnosus Bm01 removed OTA mainly by absorption through the cell wall. Furthermore, we applied L. rhamnosus Bm01 to remove OTA in grape juice or ready-to-drink coffee. Approximately 83.87% of 50 ng/mL and 100% of 20 ng/mL OTA in grape juice was removed at 48 h after incubation when treated with L. rhamnosus Bm01, respectively. Results indicated the potential of L. rhamnosus Bm01 in the decontamination of OTA in food industries.

Detection of ochratoxin A by fluorescence sensing based on mesoporous materials.

We developed a new ochratoxin A (OTA) aptamer biosensor to promptly detect OTA in food. Mesoporous silica nanoparticles were used as carriers, and aptamers were used as recognition probes and gating molecules. The fluorescent dye rhodamine 6G was loaded into mesoporous silica, and through electrostatic contact, the OTA aptamer was adsorbed on amino-modified mesoporous silica. The fluorescent dye released from the mesopore in the presence of OTA because of the conformational change induced in the aptamer by the target. The amount of ochratoxin was determined by measuring the fluorescence intensity. Our findings revealed a positive relationship between the fluorescence intensity and OTA concentration, with a limit of detection of 0.28 ng mL-1, and the detection range was 0.05-200 ng mL-1. The recovery rate was 80.7%-110.8% in real samples. The proposed approach is suitable for the quantification of other toxins.

Nanobody/NanoBiT system-mediated bioluminescence immunosensor for one-step homogeneous detection of trace ochratoxin A in food

Hazardous small molecules in food and environment seriously threatens human health, which requires sensitive and rapid tools for monitoring. Using a previously identified nanobody against ochratoxin A (OTA), we herein proposed a homogeneous sensing platform “nanobody/NanoLuc Binary Technology (NanoBiT) system” and developed a nanobody/NanoBiT system-mediated bioluminescence immunosensor (NBL-Immunosens) for OTA using LgBiT (Lg) and SmBiT (Sm), two subunits of the split nanoluciferase (NanoLuc). The core elements of NBL-Immunosens include Lg-nanobody fusion (NLg) and Sm-labeled OTA-bovine serum albumin conjugate (OSm). The antigen-antibody interaction between NLg and OSm triggers the reconstitution of NanoLuc for generating luminescence signals. Moreover, free OTA can compete with OSm for binding to NLg, resulting the decrease of dose-dependent signals. NBL-Immunosens can detect OTA in a one-step assay of 5 min without washing and exhibit a limit of detection of 0.01 ng/mL with a linear range of 0.04–2.23 ng/mL. It shows high selectivity for OTA and has good accuracy and precision in the spiking-and-recovery experiments. Furthermore, its effectiveness was evaluated with real cereal samples and confirmed by liquid chromatography tandem mass spectrometry and commercial ELISA kits. Hence, the NBL-Immunosens is a very promising tool for rapid, accurate, and selective detection of trace OTA in food.

An ultrasensitive electrochemical aptasensor based on Pd@PCN-222 as a signal probe coupled with exonuclease III-assisted cycling amplification for the detection of ochratoxin A

An ultrasensitive electrochemical aptasensor was established based on metalloporphyrin metal organic frameworks (Pd@PCN-222) with peroxidase-like activity and exonuclease III-assisted recycling magnification for the detection of ochratoxin A (OTA). Pd@PCN-222 showed well peroxidase-like activities due to Fe-tetrakis (4-carboxyphenyl) porphyrin (Fe-TCPP) as a heme-like ligand and in situ supported with palladium nanoparticle, which could be used for the label of ssDNA instead of nature enzyme. The designed exonuclease III-assisted recycling magnification further improve the sensitivity for the detection. Based on this, the proposed aptasensor showed a well linear relationship within the scope of 1 × 10−5 ng mL−1 to 10 ng mL−1 and a low detection limit of 6.79 fg mL−1. Additionally, the application in the real spiked oil samples confirmed the dependability and veracity of the aptasensor. Hence, the designed aptasensor has comprehensive application prospects for the detection of OTA in food.

Polyols Induce the Production of Antifungal Compounds by Lactobacillus plantarum.

Mycotoxins may be present in nuts, coffee, cereals, and grapes, among other products. Increasing concerns about human health and environmental protection have driven the application of biological control techniques that can inhibit fungal contaminants. In this study, the growth inhibition of the ochratoxigenic fungus Aspergillus carbonarius Ac 162 was evaluated using 5 lactic acid bacteria (LAB). The LAB studied were Lactobacillus plantarum MZ801739 (J), Lactobacillus plantarum MZ809351 (31) and Lactobacillus plantarum MZ809350 (34), isolated in the Ivory Coast, and Lactobacillus plantarum MN982928 (3) and Leuconostoc citreum MZ801735 (23), isolated in Mexico. J, 31, 34, 3 and 23 are the internal strain codes from our laboratory. LAB were cultivated in De Man, Rogosa and Sharpe (MRS) broth, and different polyols (glycerol, mannitol, sorbitol, and xylitol) were added to the culture broth to stimulate the production of antifungal compounds. The fungal inhibition studies were performed using the poisoned food technique. The highest inhibition of A. carbonarius growth was obtained by cultivating L. plantarum MZ809351 in the presence of xylitol and glycerol. Under these conditions, 1 L of the L. plantarum MZ809351 cultures were used to identify antifungal compounds. The compounds were concentrated by solid-phase extraction and then characterized by GC-MS. In addition to 9-octadecenoic acid, 3 diketopiperazines or cyclic dipeptides were identified, including cyclo (Leu-Leu), cyclo (Pro-Gly) and cyclo (Val-Phe), which were compounds related to microbial antifungal activities. Xylitol and glycerol induced the production of these antifungal compounds against A. carbonarius Ac 162. On the other hand, adding xylitol and glycerol to the MRS broth reduced the Ochratoxin A (OTA) content to 56.8 and 54.7%, respectively. This study shows the potential for using L. plantarum MZ809351 as a biocontrol agent to prevent the growth of A. carbonarius and reduce the production of OTA in foods.

Electrochemiluminescence Immunosensor Based on Nanobody and Au/CaCO3 Synthesized Using Waste Eggshells for Ultrasensitive Detection of Ochratoxin A.

A novel ultrasensitive electrochemiluminescence (ECL) immunoassay based on Au/CaCO3 was proposed for detecting ochratoxin A (OTA) in coffee. Au/CaCO3 nanocomposites synthesized using waste eggshells as the template with a large surface area and excellent electrochemical properties were applied for immobilizing a large amount of Ru(bpy)32+ and conjugating a high-affinity nanobody (prepared by the phage display technique). Coupling of the Au/CaCO3 nanocomposites and nanobody technologies provided an ultrasensitive and highly selective ECL immunosensor for OTA detection in the range of 10 pg/mL–100 ng/mL with a low detection limit of 5.7 pg/mL. Moreover, the as-prepared ECL immunosensor showed excellent performance and high stability. Finally, the proposed ECL sensor was applied to analyze OTA in coffee samples, confirming the desirable accuracy and practical applicability potential. Overall, this work presents a new nanomaterial for fabricating the sensing interface of immunosensors by harnessing natural waste as the source and a method for detecting toxic OTA in foods.

Ochratoxin A and its reaction products affected by sugars during heat processing

Ochratoxin A (OTA) is a nephrotoxin produced by many species in two fungal genera of Aspergillus and Penicillium under virtually all agricultural environments. Hence, OTA occurs frequently in agricultural commodities and their downstream products worldwide. In this study, thermal stability of OTA in the presence of sugars commonly added to food products including glucose, fructose, and sucrose was investigated by analyzing their reaction products with HPLC–FLD and LC–MS/MS. Samples were heated at three different temperatures (100, 125, and 150 °C) in 10-min intervals for up to 60 min in the absence of food matrix. Analysis showed increased OTα and OTα-amide and decreased OTA isomer (14-R-OTA) formation when OTA was heated with sugars. Among the sugars tested, adding fructose resulted in significantly lower OTA levels than glucose, sucrose, or no sugar added control. Addition of fructose also shifted OTA degradation product profile to less toxic OTα-amide, instead of OTA isomer which has similar toxicity to OTA. These results suggest that added sugars influenced the levels of OTA and its degradation products formed during thermal processing, and may provide a means to reduce the toxicity of OTA in food.