Tuning the planarity of molecularly imprinted covalent organic frameworks for selective extraction of ochratoxin A in alcohol samples.

Here, we report a monomer planarity modulation strategy for room-temperature constructing molecularly imprinted-covalent organic frameworks (MI-COFs) for selective extraction of ochratoxin A (OTA). 2,4,6-triformylphloroglucinol (Tp) was used as basic building block, while three amino monomers with different planarity were employed as modulators to explore the effect of planarity on the selectivity of MI-COFs. The MI-TpTapa constructed from Tp and the lowest planarity of monomer Tapa gave the highest selectivity for OTA, and was further used as the adsorbent for dispersed-solid phase extraction (DSPE) of OTA in alcohol samples. Coupling MI-TpTapa based DSPE with high-performance liquid chromatography allowed the matrix-effect free determination of OTA in alcohol samples with the limit of detection of 0.023 µg kg-1 and the recoveries of 91.4-97.6%. The relative standard deviation (RSD, n = 6) of intra and inter day was <3.2%. This work provides a new way to construct MI-COFs for selective extraction of hazardous targets.

Immobilization of pancreatin based on ultrasound-assisted polydopamine functionalized magnetic porous chitosan for the detoxification of ochratoxin A in wine.

Ochratoxin A (OTA) is a mycotoxin that globally contaminates fruits and their products. Since OTA have a huge negative impact on health hazards and economic losses, it is imperative to establish an effective and safe strategy for detoxification. Here, pancreatin was immobilized on the surface of polydopamine functionalized magnetic porous chitosan (MPCTS@ PDA) for the degradation of OTA. Compared with free pancreatin, MPCTS@ PDA@ pancreatin displayed excellent thermal stability, acid resistance, storage stability and OTA detoxification in wine (>58%). Moreover, the MPCTS@ PDA@ pancreatin retained 43% initial activity after 8 reuse cycles. There was no significant change in the quality of wine after MPCTS@ PDA@ pancreatin treatment. Moreover, it did not exhibit cytotoxicity which facilitated its application in wine. These results demonstrated that MPCTS@ PDA@ pancreatin can be used as a highly effective biocatalysate for OTA detoxification in wine.

[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.

Separation of ochratoxins by centrifugal partition chromatography.

The present research work was dedicated to developing an efficient method based on liquid-liquid chromatography (centrifugal partition chromatography, CPC) applicable to routine purifications of ochratoxins (OT) from the liquid culture of the strain A. albertensis SZMC 2107. The crude extract contained numerous components in addition to OTA (90.1 %,) and OTB (1.1 %,) according to HPLC examinations. For the separation of OTs by CPC, several tertiary systems based on acetonitrile, acetone, and short-chain alcohols were examined to find the most applicable biphasic system. The hexane/i-propanol/water 35:15:50 system supplemented with 0.1 % acetic acid was found to be the most efficient for use in CPC separation. Using liquid-liquid instrumental separation, the two OTs, namely OTA (2.23 mg) and OTB (0.031 mg), were successfully isolated with 96.3 % and-72.8 % purity, respectively, from 1 L ferment broth. The identities and purities of the purified components were confirmed and the performance parameters of each separation step and the whole procedure were determined. The developed method could be used effectively to purify OTs for analytical or toxicological applications.

Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

Structure-switching aptasensors for sensitive detection of ochratoxin A.

Ochratoxin A (OTA) is a toxic metabolite commonly found in various foods and feedstuffs. Accurate and sensitive detection of OTA is needed for food safety and human health. Based on a common OTA-binding aptamer (OTABA), two structure-switching OTABAs, namely OTABA4 and OTABA3, were designed by configuring a split G-quadruplex and a split G-triplex, respectively, at the two ends of OTABA to construct aptasensors for the detection of OTA. The OTABA, G-quadruplex, and G-triplex all can capture the thioflavin T (ThT) probe, thereby enhancing the fluorescence intensity of ThT. Bonding with OTA could change the conformations of OTABA and G-quadruplex or G-triplex regions, resulting in the release of the captured ThT and diminution of its fluorescence intensity. Dual conformation changes in structure-switching OTABA synergistically amplified the fluorescence signal and improved the sensitivity of the aptasensor, especially for that with OTABA3. The detection limits of the OTABA4-ThT and OTABA3-ThT systems for OTA were 0.28 and 0.059 ng ml-1 , with a 1.4-fold and 6.7-fold higher sensitivity than that of the original OTABA-ThT system, respectively. They performed well in corn and peanut samples and met the requirements of the food safety inspections.

Aptamer Molecular Beacon Sensor for Rapid and Sensitive Detection of Ochratoxin A.

Ochratoxin A (OTA) is a carcinogenic fungal secondary metabolite which causes wide contamination in a variety of food stuffs and environments and has a high risk to human health. Developing a rapid and sensitive method for OTA detection is highly demanded in food safety, environment monitoring, and quality control. Here, we report a simple molecular aptamer beacon (MAB) sensor for rapid OTA detection. The anti-OTA aptamer has a fluorescein (FAM) labeled at the 5' end and a black hole quencher (BHQ1) labeled at the 3' end. The specific binding of OTA induced a conformational transition of the aptamer from a random coil to a duplex-quadruplex structure, which brought FAM and BHQ1 into spatial proximity causing fluorescence quenching. Under the optimized conditions, this aptamer sensor enabled OTA detection in a wide dynamic concentration range from 3.9 nM to 500 nM, and the detection limit was about 3.9 nM OTA. This method was selective for OTA detection and allowed to detect OTA spiked in diluted liquor and corn flour extraction samples, showing the capability for OTA analysis in practical applications.

Mitigation potential of individual and combined dietary supplementation of local Bentonite Clay and Distillery Sludge against Ochratoxin-A induced toxicity in broilers.

BACKGROUND: This study aimed to evaluate the ameliorative effects of dietary supplementation of local bentonite clay (BN) and distillery sludge (DS) alone and in combination on ochratoxin-A (OTA) induced toxicity in broilers. For this purpose, day-old-broiler chicks (n = 270) were procured from the local market and reared under standard management conditions. After 7 days of acclimatization, birds were divided into 2 main groups A and B with respect to OTA inclusion level in feed, each with four sub-groups viz. A1-A4, each challenged with OTA at a dietary inclusion level of 250 µg/kg feed and B1-B4, each challenged with OTA at the level of 500 µg/kg feed and a common control group that was fed with basal feed throughout the experiment. In groups A and B, BN and DS were administered with feed at the rate of 10 g/kg of feed and 5 g/kg of feed alone and in combination, respectively. RESULTS: Results showed that OTA administration alone resulted in poor feed conversion ratio (FCR) and immunological responses along with increased serum levels of alanine transaminase (ALT), Aspartate transaminase (AST), urea and creatinine (P < 0.05). A significant decrease (P < 0.05) in serum protein levels (albumin, globulin and total protein) was also observed in OTA-fed groups in a dose-dependent manner. The addition of BN at 10 g/kg of OTA-contaminated feed resulted in better FCR and immunological responses as compared to those fed OTA only. The BN supplementation also conferred protection against elevation of serum biochemical parameters when compared with OTA-fed groups. However, the addition of DS could not provide significant protection (P > 0.05) on alteration of serum biochemical parameters in response to the OTA induced toxicity. The combined supplementation of BN and DS resulted in amelioration of OTA-induced toxicity and showed improved FCR, immunological, hematological and serum biochemical parameters (P < 0.05) when compared with other groups. Similarly, BN and DS resulted in a significant decline (P < 0.05) in the OTA tissue residues compared with other groups and control. CONCLUSION: In conclusion, combined dietary supplementation of BN (10 mg/kg) and DS (05 mg/kg) in feed reduced the toxic effects of OTA contamination at levels of 250 and 500 µg/kg of feed in broilers. So, the combination products of BN and DS may be successfully developed for use in poultry for protection against OTA-induced toxicity in broilers.

Hypoalbuminemia affects the spatio-temporal tissue distribution of ochratoxin A in liver and kidneys: consequences for organ toxicity.

Hypoalbuminemia (HA) is frequently observed in systemic inflammatory diseases and in liver disease. However, the influence of HA on the pharmacokinetics and toxicity of compounds with high plasma albumin binding remained insufficiently studied. The 'lack-of-delivery-concept' postulates that HA leads to less carrier mediated uptake of albumin bound substances into hepatocytes and to less glomerular filtration; in contrast, the 'concept-of-higher-free-fraction' argues that increased concentrations of non-albumin bound compounds facilitate hepatocellular uptake and enhance glomerular filtration. To address this question, we performed intravital imaging on livers and kidneys of anesthetized mice to quantify the spatio-temporal tissue distribution of the mycotoxin ochratoxin A (OTA) based on its auto-fluorescence in albumin knockout and wild-type mice. HA strongly enhanced the uptake of OTA from the sinusoidal blood into hepatocytes, followed by faster secretion into bile canaliculi. These toxicokinetic changes were associated with increased hepatotoxicity in heterozygous albumin knockout mice for which serum albumin was reduced to a similar extent as in patients with severe hypoalbuminemia. HA also led to a shorter half-life of OTA in renal capillaries, increased glomerular filtration, and to enhanced uptake of OTA into tubular epithelial cells. In conclusion, the results favor the 'concept-of-higher-free-fraction' in HA; accordingly, HA causes an increased tissue uptake of compounds with high albumin binding and increased organ toxicity. It should be studied if this concept can be generalized to all compounds with high plasma albumin binding that are substrates of hepatocyte and renal tubular epithelial cell carriers.

Isoenzyme N-Acyl-l-Amino Acid Amidohydrolase NA Increases Ochratoxin A Degradation Efficacy of Stenotrophomonas sp. CW117 by Enhancing Amidohydrolase ADH3 Stability.

Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. OTA detoxification strategies are significant to food safety. A superefficient enzyme ADH3 to OTA hydrolysis was isolated from the difunctional strain Stenotrophomonas sp. CW117 in our previous study. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA, which is an isoenzyme of ADH3. However, it is not as efficient a hydrolase as ADH3. The kinetic constant showed that the catalytic efficiency of ADH3 (Kcat/Km = 30,3938 s-1 · mM-1) against OTA was 29,113 times higher than that of NA (Kcat/Km = 10.4 s-1 · mM-1), indicating that ADH3 was the overwhelming superior detoxifying gene in CW117. Intriguingly, when gene na was knocked out from the CW117 genome, degradation activity of the Δna mutant was significantly reduced at the first 6 h, suggesting that the two enzymes might have an interactive effect on OTA transformation. Gene expressions and Western blotting assay showed that the Δna mutant and wild-type CW117 showed similar adh3 expression levels, but na deficiency decreased ADH3 protein level in CW117. Collectively, isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 but not as a dominant hydrolase for OTA transformation. IMPORTANCE Ochratoxin A (OTA) is a potent mycotoxin mainly produced by toxicogenic strains of Aspergillus spp. and seriously contaminates foods and feedstuffs. Previous OTA detoxification studies mainly focused on characterizations of degradation strains and detoxifying enzymes. Here, we identified a gene N-acyl-l-amino acid amidohydrolase NA from strain CW117, which is an isoenzyme of the efficient detoxifying enzyme ADH3. Isoenzyme NA was identified as a factor that improved the stability of ADH3 in CW117 and, thus, enhanced the degradation activity of the strain. This is the first study on an isoenzyme improving the stability of another efficient detoxifying enzyme in vivo.

Determination and detoxification of aflatoxin and ochratoxin in maize from different regions of Pakistan.

The contamination of food commodities with mycotoxins could be a serious health threat to humans and animals. Therefore, identification, quantification and reduction of mycotoxins in food commodities, particularly of aflatoxins (AFs) and ochratoxin A (OTA) in grain foods, is essentially required to guarantee safe food. This study determined the levels of AFs and OTA in 135 maize grains samples belonging to eight salient maize varieties cultivated in Pakistan, and evaluated the usefulness of radiations and adsorbents to reduce their levels. High performance liquid chromatography (HPLC)-based method was validated for the determination of AFs and OTA in maize grains. The results showed that 69 and 61% samples were positive for AFs and OTA, respectively and 54 and 22% of the respective samples had AFs and OTA above the permissible limits set by Pakistan Standards and Quality Control Authority. The concentration of AFs, AFB1and OTA in grains ranged from 14.5 to 92.4, 1.02 to 2.46 and 1.41 to 53.9 µg kg-1, respectively. Among the varieties, Pearl had the highest level of total AFs and OTA, whereas YH-5427 had the highest AFB1 level. The lowest concentration of AFs and OTA was found in Malaka and 30Y87, respectively. The use of 15 kGy gamma irradiation for 24 h, sunlight-drying for 20 h and UV irradiation for 12 h almost completely degraded the mycotoxins. The microwave heating for 120 s resulted in 9-33% degradation of mycotoxins. Moreover, the treatment of grains' extract with activated charcoal (5% w/w) removed > 96% of total AFs and AFB1, and up to 43% of OTA. The use of bentonite at the same rate removed OTA, total AFs and AFB1 by 93, 73 and 92%, respectively. Thus, it is concluded that contamination of maize grains with mycotoxins was fairly high in the collected maize grain samples in Pakistan, and treatment with radiations and adsorbents can effectively reduce mycotoxins contamination level in maize grains.

Core-shell Au@PtAg modified TiO2-Ti3C2 heterostructure and target-triggered DNAzyme cascade amplification for photoelectrochemical detection of ochratoxin A.

Efficient charge separation and utilization are critical factors to obtain a high initial signal in photoelectrochemical (PEC) aptasensor. Reports demonstrate that constructing metal/semiconductor Schottky junction can effectively improve the charge separation efficiency. Herein, a photoelectrode Au@PtAg/TiO2-Ti3C2 Schottky junction is successfully synthesized. Specifically, the Schottky junction between core-shell Au@PtAg and TiO2-Ti3C2 facilitates the efficiency of photogenerated electron transfer and enables the transfer of photogenerated electrons from TiO2-Ti3C2 to Au@PtAg. Noteworthy, the core-shell Au@PtAg acts as a photoelectron receiver to capture and store electrons, which further facilitates the separation of photogenerated electron-hole pairs, resulting enhanced photocurrent generation without sacrificial agents. Moreover, through the Mg2+-dependent DNAzyme cascade amplification, the sensitivity of the PEC aptasensor is further improved. Hence, we report an ultrasensitive PEC aptasensor for ochratoxin A (OTA) assy based on Au@PtAg/TiO2-Ti3C2 Schottky junction and Mg2+-dependent DNAzyme cascade amplification. As a result, the established PEC aptasensor exhibits excellent photocurrent performance in the range of 5 fg mL-1-10 ng mL-1 with a detection limit as low as 1.73 fg mL-1, showing high sensitivity, selectivity as well as stability. This strategy provides a versatile and promising avenue for the development of high-performance PEC aptasensor.

Structural Insights into the Mechanism of High-Affinity Binding of Ochratoxin A by a DNA Aptamer.

A 36-mer guanine (G)-rich DNA aptamer (OBA36) is able to distinguish one atomic difference between ochratoxin analogues A (OTA) and B (OTB), showing prominent recognition specificity and affinity among hundreds of aptamers for small molecules. Why OBA36 has >100-fold higher binding affinity to OTA than OTB remains a long-standing question due to the lack of high-resolution structure. Here we report the solution NMR structure of the aptamer-OTA complex. It was found that OTA binding induces the aptamer to fold into a well-defined unique duplex-quadruplex structural scaffold stabilized by Mg2+ and Na+ ions. OTA does not directly interact with the G-quadruplex, but specifically binds at the junction between the double helix and G-quadruplex through π-π stacking, halogen bonding (X-bond), and hydrophobic interaction. OTB has the same binding site as OTA but lacks the X-bond. The strong X-bond formed between the chlorine atom of OTA and the aromatic ring of C5 is the key to discriminating the strong binding toward OTA. The present research contributes to a deeper insight of aptamer molecular recognition, reveals structural basis of the high-affinity binding of aptamers, and provides a foundation for further aptamer engineering and applications.

Ochratoxin A in Slaughtered Pigs and Pork Products.

Ochratoxin A (OTA) is a mycotoxin that is produced after the growth of several Aspergillus and Penicillium spp. in feeds or foods. OTA has been proved to possess nephrotoxic, hepatotoxic, teratogenic, neurotoxic, genotoxic, carcinogenic and immunotoxic effects in animals and humans. OTA has been classified as possibly carcinogenic to humans (Group 2B) by the IARC in 2016. OTA can be mainly found in animals as a result of indirect transmission from naturally contaminated feed. OTA found in feed can also contaminate pigs and produced pork products. Additionally, the presence of OTA in pork meat products could be derived from the direct growth of OTA-producing fungi or the addition of contaminated materials such as contaminated spices. Studies accomplished in various countries have revealed that pork meat and pork meat products are important sources of chronic dietary exposure to OTA in humans. Various levels of OTA have been found in pork meat from slaughtered pigs in many countries, while OTA levels were particularly high in the blood serum and kidneys of pigs. Pork products made from pig blood or organs such as the kidney or liver have been often found to becontaminated with OTA. The European Union (EU) has established maximum levels (ML) for OTA in a variety of foods since 2006, but not for meat or pork products. However, the establishement of an ML for OTA in pork meat and meat by-products is necessary to protect human health.

Spent Coffee Grounds Valorization as Bioactive Phenolic Source Acquired Antifungal, Anti-Mycotoxigenic, and Anti-Cytotoxic Activities.

Spent coffee grounds (SCGs), which constitute 75% of original coffee beans, represent an integral part of sustainability. Contamination by toxigenic fungi and their mycotoxins is a hazard that threatens food production. This investigation aimed to examine SCGs extract as antimycotic and anti-ochratoxigenic material. The SCGs were extracted in an eco-friendly way using isopropanol. Bioactive molecules of the extract were determined using the UPLC apparatus. The cytotoxicity on liver cancer cells (Hep-G2) showed moderate activity with selectivity compared with human healthy oral epithelial (OEC) cell lines but still lower than the positive control (Cisplatin). The antibacterial properties were examined against pathogenic strains, and the antifungal was examined against toxigenic fungi using two diffusion assays. Extract potency was investigated by two simulated models, a liquid medium and a food model. The results of the extract showed 15 phenolic acids and 8 flavonoids. Rosmarinic and syringic acids were the most abundant phenolic acids, while apigenin-7-glucoside, naringin, epicatechin, and catechin were the predominant flavonoids in the SCGs extract. The results reflected the degradation efficiency of the extract against the growth of Aspergillus strains. The SCGs recorded detoxification in liquid media for aflatoxins (AFs) and ochratoxin A (OCA). The incubation time of the extract within dough spiked with OCA was affected up to 2 h, where cooking was not affected. Therefore, SCGs in food products could be applied to reduce the mycotoxin contamination of raw materials to the acceptable regulated limits.

Simple Design Concept for Dual-Channel Detection of Ochratoxin A Based on Bifunctional Metal-Organic Framework.

A simple fluorescence and electrochemical dual-channel biosensor based on bifunctional Zr(IV)-based metal-organic framework (Zr-MOF) was proposed to detect Ochratoxin A (OTA). The bifunctional Zr-MOF, with photoluminescence properties and enormous electroactive ligands, was exploited to load OTA-specific aptamers for designing signal probes, greatly simplifying the probe-fabrication process and improving sensing reliability. Upon specific recognition of aptamer toward OTA, the anchored probe was released from the sensing interface into the reaction solution. In this circumstance, the increased amount of the signal probe in reaction solution led to an enhanced fluorescence response, while the decreased amount of the signal probe on the sensing interface resulted in a diminished electrochemical response. According to the dual-channel signal change with increasing OTA concentration, the visual fluorescence strategy was established for intuitive OTA detection, and meanwhile, sensitive electrochemical assay with a detection limit of 0.024 pg/mL was also achieved with the help of one-step electrodeposition as a sensing platform. Moreover, the proposed dual-channel assay has been successfully applied to determine OTA levels in corn samples with rapid response, superior accuracy, and high anti-interference capability, providing a promising method for food safety monitoring.

A novel aptasensor based on DNA hydrogel for sensitive visual detection of ochratoxin A.

A novel visual detection mode is proposed to improve the detection sensitivity for the determination of ochratoxin A (OTA). The mode is based on aptamer recognition and the signal amplification of rolling circle amplification (RCA) products self-assembled DNA hydrogel. Moreover, gold nanoparticles (AuNPs) were directly assembled inside the DNA hydrogel by adjusting the padlock probe sequences to achieve a stronger binding force between the DNA hydrogel and AuNPs; this avoids the need for modification of AuNPs with DNA sequences. In the presence of OTA, DNA hydrogel is formed. With higher concentrations of OTA, a larger amount of DNA hydrogel is formed. When AuNPs are added to the DNA hydrogel, AuNPs can be enclosed inside the DNA hydrogel. With more DNA hydrogel, there is less AuNPs in the supernatant. Thus, the absorbance of the supernatant is anti-correlated with the concentration of OTA. After optimization of the experimental conditions, the change in the absorbance of the supernatant was linearly correlated with the concentration of OTA, in the range 0.05 to 10 ng/mL; the limit of detection was 0.005 ng/mL. The good specificity of the developed biosensor was confirmed in the presence of other mycotoxins that are coexistent with or analogues of OTA. By comparing the developed method with the ELISA method, the accuracy and stability of this new method were also verified, with good performance obtained in real samples. Diagram of the principle of the colorimetric aptasensor for OTA detection based on rolling circle amplification product self-assembled DNA hydrogel.

In situ photo-initiated polymerized oligonucleotide-functionalized hydrophilic capillary affinity monolith for highly selective in-tube microextraction of ochratoxin A mycotoxin.

A photo-initiated polymerized oligonucleotide-grafted hydrophilic affinity monolithic column was synthesized in situ, and exploited for selective in-tube solid phase micro-extraction (IT-SPME) protocol towards the sensitive detection of ochratoxin A (OTA). Only 7 min was required for the rapid polymerization of aptamer-based affinity monolith, which was much less than the reaction time of most thermal polymerization (12-16 h) and sol-gel chemistry methods (up to 52 h). Characterizations such as polymerization recipes, structure morphology, FTIR spectrum, elemental mapping, mechanical stability, and specific recognition performance were evaluated. A significantly hydrophilic nature with a low contact angle of 15° was observed, and a mixed-mode mechanism including aptamer affinity recognition and hydrophilic interaction (HI) was employed. By coupling with HPLC-fluorescence detection, the highly specific online recognition performance was achieved with an extremely low nonspecific adsorption of the analogues. The calibration curve of OTA was obtained in the concentration range 0.05-50.00 ng·mL-1 with a limit of detection (LOD, S/N = 3) of 0.012 ng·mL-1. Applied to sample analysis, acceptable recovery yields of 95.1 ± 1.4% - 99.5 ± 2.2% (n = 3) were obtained in beer and red wine. The proposed method lighted a promising way to efficiently preparing a hydrophilic aptamer-affinity monolith for highly specific recognition of trace mycotoxin by IT-SPME coupled with HPLC. A hydrophilic oligonucleotide-based affinity capillary monolith was explored via in situ photopolymerization for overcoming low preparation efficiency and achieving high-performance online IT-SPME of OTA mycotoxin.

Evaluation of fungal contamination and ochratoxin A detection in different types of coffee by HPLC-based method.

BACKGROUND: Mycotoxins are secondary fungal metabolites that are produced by some toxigenic fungi on foodstuffs which are poisoning and potentiate for human's health hazards. In coffee samples, ochratoxin A and fungal contamination were examined. METHODS: Immunoaffinity columns were used for treating of all 50 samples from four types of coffee, after that high-performance liquid chromatography was used for determining the amount of ochratoxin. For the identification of fungi, all coffee samples were cultured in appropriated media. RESULTS: The results showed that all samples were contaminated by ochratoxin A but only up to 50% of them had toxins higher than acceptable level as detected in black beans (47%), green beans (33.3%), torch (33.3%), and espresso (25%). Black coffee had a higher mean concentration of ochratoxin A than green coffee. CONCLUSION: Predominant fungi isolated from coffee samples were Aspergillus species. Finally, careful monitoring of mycotoxins in coffee samples is essential to improve the quality of this favorable beverage in future.

Ochratoxin a levels in fermented specialty coffees from Caparaó, Brazil: Is it a cause of concern for coffee drinkers?

Although postharvest coffee fruit fermentation can improve coffee flavour and quality, the mycotoxin ochratoxin A (OTA) can also be a result of microbiological activity, albeit in the later drying step of coffee processing. To evaluate the possible occurrence of OTA contamination in postharvest fruit fermentation, fourteen coffees that entailed two different postharvest fruit fermentation times were evaluated. These coffees originated in the surroundings of the village of Pedra Menina in the qualified Denomination of Origin and coffee producer region of Caparaó on the border between Minas Gerais and Espírito Santo states in Brazil. All coffees were classified according to the Specialty Coffee Association (SCA) protocol and 12 achieved specialty level. OTA was determined in all 14 coffees using immunoaffinity for sample clean-up and high-performance liquid chromatography with fluorescence detection for quantification. One sample presented an OTA concentration of 0.75 µg kg-1 and two samples showed OTA concentrations of 0.87 µg kg-1. The other samples had concentrations of OTA below the limit of quantification obtained in this work (0.64 µg kg-1). Thus, all samples showed OTA concentrations far below the most stringent maximum residue limit (MRL) of 5 µg kg-1 established for roasted coffees by European legislation. These low levels were similar to most of the previous results for Brazilian coffees listed and tabled in this work. This comparison showed that OTA contamination due to this kind of postharvest process - fruit fermentation - should not be a concern for producers and consumers of these fermented coffees.