Ochratoxin A Concentration Research Articles

The concentration and prevalence of aflatoxins and ochratoxin A in imported rice (Oryza sativa) samples from Iranian market: a probabilistic dietary risk assessment

ABSTRACT This investigation aimed to measure the concentrations of total aflatoxin (AFT), aflatoxin B1 (AFB1), and ochratoxin A (OTA) in imported rice samples to Iran and conduct a probabilistic health risk assessment for consumers. Three thousand seven hundred fifty-one imported rice samples were randomly collected from March 2022 to April 2024. The AFT, AFB1, and OTA concentrations were analysed using high-performance liquid chromatography with a flame ionisation detector (HPLC-FID) and an ultraviolet detector (HPLC-UV). The ranking of mycotoxins based on mean concentration in rice was as follows: AFT (3.996 ± 6.925 µg/kg) > AFB1 (2.648 ± 5.746 µg/kg) > OTA (0.088 ± 0.847 µg/kg). The prevalence of these mycotoxins was found to be AFT present in 897 out of 3,710 samples (24.18%), AFB1 in 657 samples (17.52%), and OTA in 40 samples (1.07%). Significant correlations were observed between AFT and AFB1 (C: 0.96, p-value < 0.001), AFT and OTA (C: 0.28, p-value < 0.001), and AFB1 and OTA (C: 0.15, p-value < 0.001). The 95th percentile of the Margin of Exposure (MOE) for AFB1 in adult and child consumers was 0.930 and 0.270, respectively, while the Hazard Quotient (HQ) for OTA in adults and children was 0.030 and 0.100, respectively. The estimated cancer risk percentages for AFB1 in rice were 22.220 for adults and 43.500 for children. Overall, consuming imported rice containing AFT, AFB1, and OTA does not pose a significant non-carcinogenic risk to consumers’ health. However, the carcinogenic risk in Iran is higher than in other countries. Therefore, it is recommended that more effective and continuous monitoring programmes be implemented to ensure the quality of imported rice.

Ochratoxin a levels in Turkish coffee: a probabilistic health risk assessment via Monte Carlo simulation.

Throughout history, Turkish coffee has been the most widely consumed type of coffee in Turkey. Therefore, the goal of this study was to determine the amount of ochratoxin A (OTA) present in Turkish coffee and to analyze any potential health hazards. A total of 41 Turkish coffees were collected and analyzed for OTA activity using a competitive enzyme immunoassay (ELISA). Furthermore, dietary exposure and health risk assessments for the Turkish population were calculated based on analytical results and coffee consumption data from the Turkish Nutrition and Health Survey 2019 (TNHS-2019) in three age categories (15-18, 19-64, and 65 + years). Nine of the samples contained more than 2.5μg/kg OTA, with an average of 5.24μg/kg. The OTA concentration in 3 samples exceeded the permissible maximum limit (5μg/kg) established by Turkish legislation, and the mean concentration was 8.41μg/kg. A margin of exposure (MOE) approach was used for risk characterization, considering both non-neoplastic and neoplastic consequences. There were no concerns about health risks because MOEs were more than 10,000 for all categories. Although the levels of OTA analyzed in Turkish coffee did not pose a risk to individuals in the three age categories, emphasis should be placed on minimizing and controlling OTA concentrations in Turkish coffee. Additionally, it is also necessary to consider other food sources that could contribute to OTA exposure.

A novel four-modal nano-sensor based on two-dimensional Mxenes and fully connected artificial neural networks for the highly sensitive and rapid detection of ochratoxin A

Timely and accurate on-site detection of ochratoxin A (OTA) is extremely important for global public health. In this study, a fluorescence/colorimetric biosensor based on Ti3C2 nano-materials (Ti3C2-NMS) and a machine-learning (ML) based fluorescence/colorimetric intelligent learning system for detection of OTA concentration (COTA) were developed. The sensor was fabricated by functionalizing Ti3C2-NMS prepared by physical-exfoliation assisted metal-ion-induction using ssDNA. The Ti3C2-NMS exhibited good fluorescence quenching characteristics (FQC) and peroxidase-like activity (PLA). More surprisingly, the functionalization of Ti3C2-NMS by ssDNA further enhanced the FQC and PLA of the material, which could be used for dual-mode detection of OTA. When different COTA existed, ssDNA competitively bound to OTA, resulting in regular changes in fluorescence and colorimetric signals of the sensor, which realized the accurate and sensitive biosensing detection of OTA in two modalities. Based on a series of fluorescent/colorimetric RGB datasets collected by a self-developed application, a dual-channel ML model had been developed. This model can be integrated into mobile phones, clouds, and PCs to achieve intelligent sensing detection of OTA with the assistance of fully connected artificial neural networks. The method constructed had high specificity, low cost, and fast responsiveness, with a LOD as low as 1.58 pg mL−1, indicating excellent potential for application and promotion.

The Effect of Synbiotics and Probiotics on Ochratoxin Concentrations in Blood and Tissues, Health Status, and Gastrointestinal Function in Turkeys Fed Diets Contaminated with Ochratoxin A.

The aim of this study was to evaluate carcass quality and analyze gastrointestinal functional status, ochratoxin A (OTA) accumulation in tissues and organs, and the health status of turkeys fed diets contaminated with OTA and supplemented with synbiotic preparations in comparison with commercial probiotic feed additives. The research involved 120 female BIG 6 turkeys, divided into six treatment groups (five replicates, four birds per replicate). Wheat naturally contaminated with OTA (662.03 μg/kg) was used in turkey diets. Turkeys in group 1 received an OTA-contaminated diet without additives. Groups 2 and 3 received 0.4 g/kg of probiotic preparation BioPlus 2B or Cylactin. Groups 4, 5, and 6 received 0.5 g/kg of synbiotics S1, S2, or S3. The following parameters were monitored: growth performance, carcass quality, gastrointestinal tract structure and digesta pH, health status, and concentrations of OTA in the blood and tissues of turkeys. The study found no significant differences in the growth performance and carcass quality of turkey. However, the introduction of probiotics or synbiotics into OTA-contaminated feed mixtures resulted in a reduced pH of the digesta in certain sections of the turkey digestive tract (p < 0.05). Additionally, the tested synbiotic additives significantly reduced liver weight in turkeys at weeks 6 and 15 (p < 0.05). The addition of probiotic and synbiotic preparations based on lactic acid bacteria strains, inulin, and S. cerevisiae yeasts to OTA-contaminated diets in commercial turkey farming may improve health status (p < 0.05) and reduce mycotoxin accumulation in organs and tissues of poultry (p < 0.05).

Reduction in Ochratoxin A Occurrence in Coffee: From Good Practices to Biocontrol Agents.

Ochratoxin A (OTA) is a mycotoxin mainly produced by Aspergillus section Circumdati and section Nigri across the coffee chain. OTA is nephrotoxic and is a threat to human health. This review summarizes current knowledge on how to reduce OTA concentration in coffee from farm to cup. After a brief introduction to the OTA occurrence in coffee, current good management practices are introduced. The core of this review focuses on biocontrol and microbial decontamination by lactic acid bacteria, yeasts and fungi, and their associated enzymes currently reported in the literature. Special attention is given to publications closest to in vivo applications of biocontrol agents and microbial OTA adsorption or degradation agents. Finally, this review provides an opinion on which future techniques to promote within the coffee supply chain.

SERS aptasensor for simultaneous detection of ochratoxin A and zearalenone utilizing a rigid enhanced substrate (ITO/AuNPs/GO) combined with Au@AgNPs

The contamination of mycotoxins poses a serious threat to global food security, hence the urgent need for simultaneous detection of multiple mycotoxins. Herein, two SERS nanoprobes were synthesized by embedded SERS tags (4-mercaptopyridine, 4MPy; 4-mercaptobenzonitrile, TBN) into the Au and Ag core–shell structure, and each was coupled with the aptamers specific to ochratoxin A (OTA) and zearalenone (ZEN). Meanwhile, a rigid enhanced substrate Indium tin oxide glass/AuNPs/Graphene oxide (ITO/AuNPs/GO) was combined with aptamer functionalized Au@AgNPs via π-π stacking interactions between the aptamer and GO to construct a surface-enhanced Raman spectroscopy (SERS) aptasensor, thereby inducing a SERS enhancement effect for the effective and swift simultaneous detection of both OTA and ZEN. The presence of OTA and ZEN caused signal probes dissociation, resulting in an inverse correlation between Raman signal intensity (1005 cm−1 and 2227 cm−1) and the concentrations of OTA and ZEN, respectively. The SERS aptasensor exhibited wide linear detection ranges of 0.001–20 ng/mL for OTA and 0.1–100 ng/mL for ZEN, with low detection limits (LOD) of 0.94 pg/mL for OTA and 59 pg/mL for ZEN. Furthermore, the developed SERS aptasensor demonstrated feasible applicability in the detection of OTA and ZEN in maize, showcasing its substantial potential for practical implementation.

A visual biosensor chip based on the enhanced electrochemiluminescence with low triggered potential for ochratoxin A detection

The introduction of a coreaction accelerator can effectively enhance the electrochemiluminescence (ECL) response. However, the development of coreaction accelerators for ruthenium (Ru) complex, one of the most widely used ECL emitters, is still in its early stages. In this study, the application of BiOI as a multifunctional coreaction accelerator for the Ru/N-Butyldiethanolamine (DBAE) system for the first time. The BiOI microspheres not only exhibit a fivefold enhancement in ECL signal but also reduce the trigger potential, potentially mitigating interference factors associated with high applied voltage. The nanoflower structure of BiOI microspheres also facilitates the immobilization of Ru(bpy)32+. Building upon this, a portable visual biosensor chip was developed for the detection of ochratoxin A (OTA). The biosensor chip comprises a sensing cell and a reporting cell. When OTA toxins are detected in the microenvironment, the aptamers attached to the surface of the sensing cell are released, resulting in a decrease in impedance on the electrode surface. Consequently, the ECL emission from Ru(bpy)32+/BiOI microspheres modified onto the reporting cell is enhanced. The concentration of OTA can be determined by analyzing the intensity change of ECL imaging, eliminating the need for specific instruments. This visual ECL biosensor chip offers the advantages of portability, ease of operation, low cost, and demonstrates excellent performance in detecting beer samples.

Aspergillus niger Ochratoxinase Is a Highly Specific, Metal-Dependent Amidohydrolase Suitable for OTA Biodetoxification in Food and Feed.

Microbial enzymes can be used as processing aids or additives in food and feed industries. Enzymatic detoxification of ochratoxin A (OTA) is a promising method to reduce OTA content. Here, we characterize the full-length enzyme ochratoxinase (AnOTA), an amidohydrolase from Aspergillus niger. AnOTA hydrolyzes OTA and ochratoxin B (OTB) mycotoxins efficiently and also other substrates containing phenylalanine, alanine, or leucine residues at their C-terminal position, revealing a narrow specificity profile. AnOTA lacks endopeptidase or aminoacylase activities. The structural basis of the molecular recognition by AnOTA of OTA, OTB, and a wide array of model substrates has been investigated by molecular docking simulation. AnOTA shows maximal hydrolytic activity at neutral pH and high temperature (65 °C) and retained high activity after prolonged incubation at 45 °C. The reduction of OTA levels in food products by AnOTA has been investigated using several commercial plant-based beverages. The results showed complete degradation of OTA with no detectable modification of beverage proteins. Therefore, the addition of AnOTA seems to be a useful procedure to eliminate OTA in plant-based beverages. Moreover, computational predictions of in vivo characteristics indicated that AnOTA is neither an allergenic nor antigenic protein. All characteristics found for AnOTA supported the suitability of its use for OTA detoxification in food and feed.

Effects of fermentation time, baking, and storage on ochratoxin A levels in sourdough flat bread.

Ochratoxin A (OTA), which is one of the most important mycotoxins in terms of human health, can be found in cereal products such as bread, "bazlama" (traditional flatbread), and pita bread, as well as cereals such as wheat, barley, and corn. This study aimed to determine the effect of different fermentation times, baking, and storage for various periods on the presence of OTA in sourdough bazlama. Bazlama flour was contaminated with OTA concentrations of 5 and 10 μg/kg. After two different fermentation times (1.5 and 3 h), baking at 300 ± 5°C, and storage at room temperature (25 ± 2°C) for 0, 5, and 10 days, the change in OTA levels of bazlama samples was determined by the high-performance liquid chromatography with fluorescence detector (HPLC-FLD) method. The effect of different storage periods on the presence of OTA is insignificant. Although a general decrease in OTA level has been determined, it has been found that long-term fermentation (at least 3 h) was more effective, especially in flours with a high concentration (10 μg/kg) of OTA contamination. It has been determined that bazlama made from contaminated flours with OTA levels of 5 and 10 μg/kg contained OTA levels exceeding 3 μg/kg when long-term fermentation was not used. This is the maximum permitted limit set by the Turkish Food Codex and the European Commission, indicating that it is not suitable for consumption in this position.

Development of a rapid method for determination of OchratoxinA in grape mash and wine.

The occurrence of Ochratoxin A (OTA) in wine is commonly known, but there is only limited information about its occurrence in grape mash and wines of German origin. Climate change has led to higher temperatures in the southern regions of Germany, which may increase the growth of fungi associated with the production of OTA and increase the content of this mycotoxin in grapes. A safe and rapid UHPLC-FLD method was developed and validated to assess the contamination of grape mash and wine with OTA. A total of 71 samples of grape mash and 30 wines from various wine producers in Baden-Württemberg, Germany, were analysed for OTA content. The results showed that no samples contained OTA in concentrations above the limit of detection. Further monitoring of samples from different vintages is needed.

Occurrence of ochratoxin A in cocoa beans and bean-to-bar chocolates.

The growing health consciousness of consumers has led to an increase in the consumption of artisanal chocolates, mainly due to their recognized health benefits. However, processing steps such as fermentation and drying of cocoa beans can favor the growth of ochratoxigenic fungi. This study aimed to assess the occurrence of ochratoxin A (OTA) in cocoa beans (purchased from e-commerce and post-harvest processing) and bean-to-bar chocolates sold in Brazil. An HPLC-FLD method was validated, with recovery values between 84 and 97% and limits of detection and quantification of 0.04 and 0.01 µg/kg, respectively. OTA was detected in 30% of the cocoa bean samples studied (n = 43), with values ranging from < 0.04 to 1.18 µg/kg. Regarding the bean-to-bar chocolates (n = 62), the OTA concentrations ranged from < 0.04 to 1.11 µg/kg, with a prevalence in semi-sweet and dark chocolates. Despite representing a growing market, to the best of our knowledge, this is the first study to report OTA concentrations in bean-to-bar chocolates and Brazilian cocoa beans used to produce this type of chocolate.

Evaluating the combined and individual cytotoxic effect of beauvericin, enniatin B and ochratoxin a on breast cancer cells, leukemia cells, and fresh peripheral blood mononuclear cells

Beauvericin (BEA), Enniatin B (ENN B), and Ochratoxin A (OTA) are mycotoxins produced by fungi species. Their main effect on several organs and systems is associated with chronic exposure going from immunotoxicity, estrogenic disorders, and renal failure to cancer (in animals and humans). OTA belongs to Group 1 according to the International Agency for Research in Cancer (IARC) and it has legislated limited values; not happening for BEA nor ENN B. Exposure to mixtures of mycotoxins occurs through food intake in daily consumption. The aim of this study was to evaluate the implication of BEA, ENN B, and OTA individually and combined in producing cytotoxicity in cells for immunological studies and cancer cell lines (human leukemia cells (HL-60), fresh human peripheral blood mononuclear cells (PBMCs), and human breast cancer (MDA-MB-231) cells). Cells were treated for 4 h and 24 h at different concentrations of BEA, ENN B, and OTA, respectively. Viability assays were carried out by flow cytometry using DAPI (4′,6-diamindino-2-phenylindole, dihydrochloride) as a viability dye and the potential effects of synergism, addition, and antagonism were assessed through the Chou and Talalay method. Individual OTA treatment exerted the greatest cytotoxicity for PBMC cells (IC50 0.5 μM) while ENN B for HL-60 (IC50 0.25 μM) and MDA-MB-231 (IC50 0.15 μM). In binary combination [ENN B + OTA] resulted in exerting the greatest cytotoxicity for HL-60 and MDA-MB-231 cells; while [BEA + OTA] in PBMC cells. The triple combination resulted in being highly cytotoxic for PBMC cells compared to HL-60 and MDA-MB-231 cells. In summary, PBMC cells were the most sensible cells for all three mycotoxins and the presence of OTA in any of the combinations had the greatest toxicity causing synergism as the most common cytotoxic effect.

Study the Correlation of Ochratoxin A with a Prevalence of Breast Cancer in Al-Najaf Province, Iraq

Abstract Background: Breast cancer is a prevalent disease affecting numerous individuals worldwide. This study focuses on investigating the concentration of Ochratoxin A (OTA) in female breast cancer patients in the Al-Najaf Province. Previous research suggests a potential correlation between OTA and breast cancer. Objectives: The objective of this study was to determine the concentration of OTA and its association with breast cancer in females in the Al-Najaf Province. Materials and Methods: A total of 40 female breast cancer patients who received treatment at The National Educational Oncology Hospital in the Al-Najaf Province from April to August 2022 were included in this study. Additionally, 20 healthy females served as the control group. Serum samples were collected in sterile tubes (Eppendorf tube) and analyzed using high-performance liquid chromatography (HPLC) to quantitatively diagnose the OTA ratio. Patient information, including age, was recorded in a data sheet. Results: The results demonstrated a significant increase (P &lt; 0.05) in OTA concentrations among the total number of breast cancer patients compared to the control group. The concentrations of OTA were measured at 0.2720 and 0.2186 ng/mL in breast cancer patients, indicating a higher OTA level in this group. Conclusion: This study provides evidence of a significant association between OTA concentration and breast cancer in female patients from the Al-Najaf Province. These findings contribute to the understanding of the role of OTA in breast cancer development. Further research is warranted to explore the mechanisms underlying this relationship and potentially develop targeted interventions.

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.

A photothermal aptasensor based on rolling circle amplification-enriched DNAzyme for portable detection of ochratoxin A in grape juice

Aptasensors for detection of ochratoxin A (OTA) have been extensively studied, but the majority of them require costly and large-scale equipment as signal readers. Herein, a photothermal aptasensor capable of portable detection of OTA through a thermometer was developed on basis of aptamer structural switching and rolling circle amplification (RCA)-enriched DNAzyme. Oligonucleotides and alkaline phosphatase (ALP) modified magnetic beads were prepared. The binding of aptamers to OTA led to the release of ALP labeled complementary DNA. After magnetic separation, ALP catalyzed the padlock dephosphorylation, inhibiting the subsequent RCA reaction. This process converted the OTA concentration into the amount of the photothermal reagent oxTMB produced from the catalytic reaction induced by RCA-enriched DNAzyme. Under the optimal conditions, the detection limit (LOD) of this aptasensor was 2.28 nM in a clean buffer, while the LOD reached 2.43 nM in 2 % grape juice. The good performance of the photothermal aptasensor makes it possible to measure OTA pollution in low resource environments.

Mycotoxin contamination in organic and conventional cereal grain and products: A systematic literature review and meta-analysis.

There is still considerable controversy about the relative risk of mycotoxin exposure associated with the consumption of organic and conventional cereals. Using validated protocols, we carried out a systematic literature review and meta-analyses of data on the incidence and concentrations of mycotoxins produced by Fusarium, Claviceps, Penicillium, and Aspergillus species in organic and conventional cereal grains/products. The standard weighted meta-analysis of concentration data detected a significant effect of production system (organic vs. conventional) only for the Fusarium mycotoxins deoxynivalenol, with concentrations ∼50% higher in conventional than organic cereal grains/products (p<0.0001). Weighted meta-analyses of incidence data and unweighted meta-analyses of concentration data also detected small, but significant effects of production system on the incidence and/or concentrations of T-2/HT-2 toxins, zearalenone, enniatin, beauvericin, ochratoxin A (OTA), and aflatoxins. Multilevel meta-analyses identified climatic conditions, cereal species, study type, and analytical methods used as important confounding factors for the effects of production system. Overall, results from this study suggest that (i) Fusarium mycotoxin contamination decreased between the 1990s and 2020, (ii) contamination levels are similar in organic and conventional cereals used for human consumption, and (iii) maintaining OTA concentrations below the maximum contamination levels (3.0μg/kg) set by the EU remains a major challenge.

Real-time, ultra-sensitive and label-free detection of OTA based on DNA aptamer functionalized carbon nanotube field-effect transistor

Ochratoxin A (OTA), widely recognized as a mycotoxin contaminant across various food products, poses significant health risks attributed to its nephrohepatotoxic and carcinogenic effects. Consequently, detecting OTA is crucial for ensuring food safety and preventing mycotoxicosis. In our study, we employed a carbon nanotube field-effect transistor (CNT FET) functionalized with DNA aptamers for targeted OTA detection. The sensor achieved a remarkably low detection threshold of 0.2 femtomolars (fM) in phosphate-buffered saline (PBS), enabling real-time detection of OTA over a broad concentration spectrum spanning from 8 fM to 80 pM. This research confirmed the sensor’s superior selectivity by effectively distinguishing OTA from non-target fungal toxins and showcased its rapid analysis capabilities across diverse complex matrices. Utilizing homogenized liquids from ten representative food samples for analysis, the sensor delivered real-time responses within 100 s, accurately identifying varying OTA concentrations down to 80 fM. In conclusion, this biosensor provides a label-free, highly sensitive, and rapid detection method for OTA, introducing novel solutions for effective mycotoxin surveillance in food safety. This biosensor also paves the way for the creation of a multiplex platform for concurrent monitoring of multiple fungal toxins.

A fluorescence and colorimetric dual-mode immunoassay for detection of ochratoxin A based on cerium nanoparticles

Ochratoxin A (OTA) is a widely distributed mycotoxin that seriously endangers food safety and human health. Therefore, establishing a sensitive and simple method for detecting OTA is crucial to ensure food safety. A fluorescence and colorimetric dual-mode immunoassay was established using cerium-based nanoparticles CPNs(Ⅳ) for detection of OTA. CPNs(IV) with good oxidase activity can catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to produce visible blue color signal. Under the catalysis of ALP, the ascorbic acid 2-phosphate (AA2P) was hydrolyzed to form ascorbic acid (AA), which reduced CPNs(Ⅳ) to CPNs(III). The produced CPNs(III) can emit significant fluorescence signal. Because the concentration of OTA was inversely proportional to the concentration of IgG-ALP in the microplate, the fluorescence signal was gradually decreased and the blue color signal gradually increased with the addition of OTA. The limits of detection (LODs) of this dual-mode immunoassay were 0.404 ng/mL (for fluorescent mode) and 0.962 ng/mL (for colorimetric mode). The recoveries of OTA were 97.60 %–103.55 % for the fluorescence mode and 99.12 %–102.60 % for the colorimetric mode in corn samples. These results indicated the dual-mode immunoassay was sensitive and reliable for detection of OTA in actual samples.

A fluorescence method based on malachite green/aptamer for detection of ochratoxin A in traditional Chinese medicines

A label-free fluorescence method based on malachite green/aptamer was developed for the detection of ochratoxin A(OTA) in traditional Chinese medicines. Malachite green itself exhibits weak fluorescence. Upon interaction with the aptamer specific to OTA, the G-quadruplex structure of the aptamer provides a protective microenvironment for malachite green, which significantly enhances its fluorescence signal. After OTA is added, preferential binding occurs between the aptamer and OTA, and malachite green will be released from the aptamer, which weakens the fluorescence signal. According to this principle, this paper established a fluorescence method with the aptamer of OTA as the recognition element and malachite green as the fluorescent probe for the detection of OTA in traditional Chinese medicines. The key experimental factors such as the concentrations of metal ions, aptamer, and malachite green were optimized to improve the performance of the method. OTA was detected under the optimal experimental conditions, and the results showed that with the increase in OTA concentration, the fluorescence signal gradually weakened. Within the range of 20-1 000 nmol·L~(-1), the OTA concentration was linearly correlated with the fluorescence signal ratio ΔF/F(ΔF=F_0-F, where F_0 is the fluorescence signal of aptamer/malachite green, and F is the fluorescence signal of OTA/aptamer/malachite green), with R~2 of 0.995. The limit of detection of the established method was 7.1 nmol·L~(-1). Furthermore, three substances structurally similar to OTA and two mycotoxins that may coexist with OTA were selected for experiments, which aimed to examine the cross-reactivity and specificity of the established method. The cross-reactivity experiments demonstrated that the interferers did not significantly affect the fluorescence signal of the detection system. The specificity experiments revealed that when mycotoxins were mixed with OTA, the fluorescence signal generated by the mixture closely resembled that of OTA itself. The results indicated that even in the presence of interferents, the established method remained unaffected and demonstrated excellent specificity. Additionally, this method exhibited remarkable reproducibility and stability. In the case of simple centrifugation and dilution of traditional Chinese medicine samples(Puerariae Lobatae Radix, Sophorae Flavescentis Radix, and Periplocae Cortex), the OTA detection method was applicable, with recovery rates ranging from 91.5% to 121.3%. Notably, this approach does not need complex pretreatment of traditional Chinese medicines while offering simple operation, low detection costs, and short detection time. Furthermore, by incorporating aptamers into the quality evaluation of traditional Chinese medicines, this method expands the application scope of aptamers.

Occurrence of ochratoxin A in Sichuan bacon from different geographical regions and characterization and biocontrol of ochratoxigenic Aspergillus westerdijkiae strain 21G2-1A

Sichuan bacon represents the most prevalent dry-cured meat product across Southwest China, but it is vulnerable to fungal spoilage. In the present study, a total of 47 Sichuan bacons were obtained from different regions of the Sichuan Province and analyzed for the presence of ochratoxin A (OTA), yielding a positive rate of 23.4 % (11/47). All the observed OTA concentrations exceeded the maximum admissible dose in meat products (1 μg/kg) established by some EU countries, with the highest OTA concentration being 250.75 μg/kg, which raises a food safety concern and reveals the need for a standardized scientific processing protocol. Then, an OTA-producing fungus named 21G2-1A was isolated from positive samples and found to be Aspergillus westerdijkiae. Further characterization suggested a positive correlation between fungal growth and OTA production. The optimal temperature for the former was 25 °C, while it was 20 °C for the latter. Although the A. westerdijkiae strain 21G2-1A demonstrated greater mycelium growth in the presence of NaCl, OTA production was significantly dismissed when the salinity was greater than 5 %. Four lactic acid bacteria (LAB) were screened out as antagonists against the ochratoxigenic fungus. In vitro evaluation of the antagonists revealed that live cells inhibited fungal growth, and adsorption also contributed to OTA removal at different levels. This study sheds some light on OTA control in Sichuan bacon through a biological approach.