For the US Food and Drug Administration\'s (FDA) mycotoxin program, the desired application of certified reference materials (CRMs) is primarily for validating methods for the determination of mycotoxins in foods and establishing metrological traceability of measurement results generated by such validated methods. The latter has been an important but insufficiently addressed challenge due to the lack of appropriate protocols and CRMs. Taking advantage of two recently available mycotoxin CRMs, OTAN-1 and Standard Reference Material (SRM) 1565, a protocol was developed for systematically examining uncertainty and establishing metrological traceability of measurement results of ochratoxin A (OTA) in corn through a series of calibration operations using the two CRMs. Instrument and method calibrations were performed using OTAN-1 and SRM 1565. The OTA value and its standard and expanded (k = 2, approximately 95% confidence) uncertainties were estimated from the calibration data and documented. These results demonstrate that the major contributing source of uncertainty is the sample matrix, highlighting the important role of the certified matrix reference material in method calibration. The value of OTA (38.5 ± 7.2 µg/g; 95% confidence interval) in the incurred sample was metrologically traceable to the International System of Units through two CRMs using the multi-laboratory validated liquid chromatography-mass spectrometry FDA compendial method. In addition, an alternative estimation of uncertainty was conducted using a one-point calibration, resulting in comparable uncertainty.

Short oligonucleotides are widely used for the construction of aptamer-based sensors and logical bioelements to modulate aptamer-ligand binding. However, relationships between the parameters (length, location of the complementary region) of oligonucleotides and their influence on aptamer-ligand interactions remain unclear. Here, we addressed this task by comparing the effects of short complementary oligonucleotides (ssDNAs) on the structure and ligand-binding ability of an aptamer and identifying ssDNAs\' features that determine these effects. Within this, the interactions between the OTA-specific G-quadruplex aptamer 1.12.2 (5\'-GATCGGGTGTGGGTGGCGTAAAGGGA GCATCGGACA-3\') and 21 single-stranded DNA (ssDNA) oligonucleotides complementary to different regions of the aptamer were studied. Two sets of aptamer-ssDNA dissociation constants were obtained in the absence and in the presence of OTA by isothermal calorimetry and fluorescence anisotropy, respectively. In both sets, the binding constants depend on the number of hydrogen bonds formed in the aptamer-ssDNA complex. The ssDNAs\' having more than 23 hydrogen bonds with the aptamer have a lower aptamer dissociation constant than for aptamer-OTA interactions. The ssDNAs\' having less than 18 hydrogen bonds did not affect the aptamer-OTA affinity. The location of ssDNA\'s complementary site in the aptamer affeced the kinetics of the interaction and retention of OTA-binding in aptamer-ssDNA complexes. The location of the ssDNA site in the aptamer G-quadruplex led to its unfolding. In the presence of OTA, the unfolding process was longer and takes from 20 to 70 min. The refolding in the presence of OTA was possible and depends on the length and location of the ssDNA\'s complementary site. The location of the ssDNA site in the tail region led to its rapid displacement and wasn\'t affecting the G-qaudruplex\'s integrity. It makes the tail region more perspective for the development of ssDNA-based tools using this aptamer.

Herein, a label-free aptasensor was designed through forming a double-stranded DNA skeleton on the glass substrate for ultrasensitive quantification of ochratoxin A (OTA) as a case study. The function fundament of the dual-responsive aptasensor was the perturbation of the vertical alignment of the liquid crystals (LCs) and intercalation of the SYBR Green I (SGI) dye molecules between the base pairs of the double-stranded DNA structure. The presence of OTA decomposed the double-stranded structure of DNA by releasing the OTA-specific aptamer from the sensing platform that induced an apparent alteration of the optical and fluorescent responses. The aptasensor specifically detected the ultra-low levels of OTA as 47.0E-9 pM (0.047 aM) and 34.0E-3 pM (34 fM) based on the polarized and fluorescent responses, respectively. The aptasensor monitored OTA in the coffee and grape drink samples. The aptasensor provides promising insight for manufacturing real-time, cost-effective, and portable sensing devices for food control usage.

Drying optimization, to mitigate fungal growth and Ochratoxin A (OTA) contamination is a key topic for raisin and currant production. Specific indicators of environmental conditions and drying properties were analyzed using two seedless grape varieties (Crimson-red and Thompson-white), artificially inoculated with Aspergillus carbonarius under open air and tunnel drying. The air temperature (T), relative humidity, grape surface temperature (Ts) and water activity throughout the drying experiment, the grapes\' moisture content and the fungal colonization and OTA contamination during the drying process and their interactions were recorded and critically analyzed. Drying properties such as the water diffusivity (Deff) and peel resistance to water transfer were estimated. The grapes Ts was 5-7 °C higher in tunnel vs. open air-drying; the infected grapes had higher maximum Ts vs. the control (around 4-6 °C). OTA contamination was higher in tunnel vs. open air-dried grapes, but fungal colonies showed the opposite trend. The Deff was higher in tunnel than in the open air-drying by 54%; the infected grapes had more than 70% higher Deff than the control, differences explained by factors affecting the water transport. This study highlighted CFU and OTA indicators that affect the water availability between red and white grapes during open air and tunnel drying, estimated by the Deff and peel resistance. This raises new issues for future research.

 Fungi are distributed worldwide and can be found in various foods and feedstuffs from almost every part of the world. Mycotoxins are secondary metabolites produced by some fungal species and may impose food safety risks to human health. Among all mycotoxins, aflatoxins (AFs), ochratoxin A (OTA), trichothecenes, deoxynivalenol (DON and T-2 toxin), zearalenone (ZEN), and fumonisins (FMN) have received much attention due to high frequency and severe health effects in humans and animals. Malaysia has heavy rainfall throughout the year, high temperatures (28 to 31 °C), and high relative humidity (70% to 80% during wet seasons). Stored crops under such conditions can easily be contaminated by mycotoxin-producing fungi. The most important mycotoxins in Malaysian foods are AFs, OTA, DON, ZEN, and FMN that can be found in peanuts, cereal grains, cocoa beans, and spices. AFs have been reported to occur in several cereal grains, feeds, nuts, and nut products consumed in Malaysia. Spices, oilseeds, milk, eggs, and herbal medicines have been reported to be contaminated with AFs (lower than the Malaysian acceptable level of 35 ng/g for total AFs). OTA, a possible human carcinogen, was reported in cereal grains, nuts, and spices in Malaysian market. ZEN was detected in Malaysian rice, oat, barley, maize meal, and wheat at different levels. DON contamination, although at low levels, was reported in rice, maize, barley, oat, wheat, and wheat-based products in Malaysia. FMN was reported in feed and some cereal grains consumed in Malaysia. Since some food commodities are more susceptible than others to fungal growth and mycotoxin contamination, more stringent prevention and control methods are required.

Aptamers are single-stranded oligonucleotides selected by SELEX (Systematic Evolution of Ligands by EXponential Enrichment) able to discriminate target molecules with high affinity and specificity, even in the case of very closely related structures. Aptamers have been produced for several targets including small molecules like mycotoxins; however, the high affinity for their respective target molecules is a critical requirement. In the last decade, the screening through computational methods of aptamers for their affinity against specific targets has greatly increased and is becoming a commonly used procedure due to its convenience and low costs. This paper describes an in-silico approach for rapid screening of ten ssDNA aptamer sequences against fumonisin B1 (FB1, n = 3), aflatoxin B1 (AFB1, n = 2) and ochratoxin A (OTA, n = 5). Theoretical results were compared with those obtained by testing the same aptamers by fluorescent microscale thermophoresis and by magnetic beads assay for their binding affinity (KD) revealing a good agreement.

The exposure and risk characterization of lactating women to aflatoxins (AFs), fumonisins (FBs), ochratoxin A (OTA) and zearalenone (ZEN) due to consumption of different types of food products in Pirassununga, São Paulo, Brazil, was assessed. Lactating women (N = 74) provided samples of foods stored and available at their households between April-August/2018, totaling 184 samples. Mycotoxins were determined in food samples by liquid chromatography-tandem mass spectrometry. According to findings, 20% (n = 36) of all food samples were contaminated with AFs at median concentrations ranging from 9.2 to 18.5 µg/kg, while OTA was detected only in three samples (rice, bread and pasta) at concentrations of 22.3, 23.8 and 48.7 µg/kg, respectively. ZEN was detected in 34 samples (18%) at median levels of 62-195 µg/kg, and FBs at median levels of 58-1546 µg/kg was observed in 22 samples (12%). Moreover, the concentration of AFs, OTA, ZEN and FBs exceeded their respective maximum permitted levels in 11 (6%), 3 (2%), 8 (4%) and 5 (3%) from total samples, respectively. Twenty-eight samples (15%) were contaminated with two or three types of mycotoxins. Corn products contributed for the highest mean probable daily intakes (PDI) of AFs (0.119 ± 0.193 µg/kg body weight (bw)/day), ZEN (0.325 ± 0.097 µg/kg bw/day) and FBs (2.936 ± 1.541 µg/kg bw/day), while wheat-based products contributed for the highest PDI of OTA (0.035 ± 0.028 µg/kg bw/day). The Margin of Exposure (MoE) value for AFs (3.72) demonstrated a high cancer risk (MoE < 10,000), and the Hazard Quotient (HQ) obtained for OTA (24.66), ZEN (4.24) and total FBs (5.01) also resulted in a non-tolerable risk (HQ > 1) via consumption of the investigated food products. Results of this trial indicate high exposure levels of lactating women to dietary mycotoxins in the studied area, which warrant concern about the possible transfer of residual mycotoxins into breast milk.

This study aimed to determine the effect of gamma radiation on the preservation of phenolic compounds and on decontamination of dry herbs in terms of ochratoxin A (OTA) and aflatoxin B1 (AFB1), using Aloysia citrodora Paláu as a case study. For this purpose, artificially contaminated dry leaves were submitted to gamma radiation at different doses (1, 5, and 10 kGy; at dose rate of 1.7 kGy/h). Phenolic compounds were analysed by HPLC-DAD-ESI/MS and mycotoxin levels were determined by HPLC-fluorescence. Eleven phenolic compounds were identified in the samples and despite the apparent degradation of some compounds (namely verbasoside), 1 and 10 kGy doses point to a preservation of the majority of the compounds. The mean mycotoxin reduction varied between 5.3% and 9.6% for OTA and from 4.9% to 5.2% for AFB1. It was not observed a significant effect of the irradiation treatments on mycotoxin levels, and a slight degradation of the phenolic compounds in the irradiated samples was observed.