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The presence of Aspergillus and Penicillium species during the fermentation process of rice wine is inevitable. The toxic secondary metabolites produced by the two fungi need to be practically monitored for rice wine safety. In this study, we developed a fast and simple vortex-assisted dispersive liquid-liquid microextraction (VADLLME) for the extraction and preconcentration of major Aspergillus and Penicillium mycotoxins (aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, mycophenolic acid, sterigmatocystin and verruculogen) in rice wine. The key parameters affecting the extraction efficiency of VADLLME procedure (pH value, type and volume of extraction and dispersive solvents) were optimized by response surface methodology (RSM) combined with Box-Behnken design (BBD) and desirability function. Target compounds were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Under the optimum conditions, good linearity ( R 2 >0.99) was achieved for all the analytes in the concentration range of 0.5–100 μg/L. The recoveries for spiked samples at three concentration levels were between 84.5% and 111.2%, with the relative standard deviations (RSD) lower than 20%. The limits of detection (LOD) and quantification (LOQ) ranged from 0.05 to 0.5 μg/L and from 0.1 to 1.0 μg/L, respectively. Simplicity in operation, low cost and low usage of organic solvents were the major advantages of the developed method. The method was successfully applied to the analysis of rice wine samples. It was found that mycophenolic acid had high incidence rate in rice wine, which needed to be continuously monitored. [ABSTRACT FROM AUTHOR]

The primary objective of this study was to synthesize three types of cross-linked chitosan polymers and further investigate their adsorption capability for multiple mycotoxins, including aflatoxin B 1 (AFB 1 ), ochratoxin A (OTA), zearalenone (ZEN), fumonisin B 1 (FB 1 ), deoxynivalenol (DON) and T-2 toxin (T2). Among these synthetic adsorbents, cross-linked chitosan-glutaraldehyde complex presented the highest adsorption capability for AFB 1 (73%), OTA (97%), ZEN (94%) and FB 1 (99%), but no obvious adsorption for DON and T2 (<30%). The effect of various incubation conditions (contact time, dosage and pH) was also studied. Subsequently, the experimental data were fitted to Langmuir, Freundlich and Hill models. The best fitting model to describe AFB 1 and FB 1 adsorption was Langmuir model ( R 2 ≥ 0.99), with the theoretical maximum adsorption amounts of 5.67 mg/g for AFB 1 and 15.7 mg/g for FB 1 . The Hill model was the best model for OTA and ZEN adsorption ( R 2 > 0.98), with the predicted maximum adsorption amounts were 24.8 mg/g for OTA and 9.18 mg/g for ZEN. In addition, the adsorption capability of adsorbent for the simultaneous presence of multiple mycotoxins was also evaluated in buffer system and simulated gastrointestinal condition. The results indicated that the coexisted multiple mycotoxins didn't affected the adsorption capability of adsorbent, whereas the adsorption amounts of toxins were decreased by some gastrointestinal components. The findings of this research suggest that chitosan–glutaraldehyde complex has the potential to be applied as multitoxin adsorbent material for reducing the combined adverse effect of multiple mycotoxins on humans and animals. [ABSTRACT FROM AUTHOR]

In this study, a reliable and fast method using a quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction procedure without any clean-up step was developed for simultaneous extraction of 15 mycotoxins, i.e., aflatoxin B1, aflatoxin B2, aflatoxin G1, aflatoxin G2, aflatoxin M1, aflatoxin M2, deoxynivalenol, 3-acetyldeoxynivalenol, 15-acetyldeoxynivalenol, de-epoxy-DON, zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, and β-zearalanol, from eggs. High-performance liquid chromatography tandem mass spectrometry was used to separate and detect all of the analytes. Electrospray ionization at both negative and positive modes and multiple reaction-monitoring mode were applied to detect these analytes. The main factors, such as extraction time, extraction solvent, evaporation temperature, and pH of the solvent, were carefully optimized to improve the extraction efficiency. The coefficients of determination of the calibration curves ranged from 0.9884 to 0.9998. The recoveries of most of the analytes were between 71.3% and 105.4% at three concentration levels, except for AFB1 that showed recovery rates of not more than 67.5% in all concentrations. The repeatability and intra-lab reproducibility of this method were both lower than 15% and 25%, respectively. The limit of quantification ranged from 0.2 μg/kg to 5 μg/kg. The matrix effect was evaluated and reduced by the use of matrix-matched calibration curves. The validated method was applied in a pilot study to analyze mycotoxin contamination in 12 eggs, and trace amounts of deoxynivalenol, 15-acetyldeoxynivalenol, aflatoxin B1, aflatoxin G2, zearalenone and β-zearalenol were detected in these samples. [ABSTRACT FROM AUTHOR]

Mycotoxins have the potential to enter the human food chain through carry-over of contaminants from feed into animal-derived products. The objective of the study was to develop a reliable and sensitive method for the analysis of 30 mycotoxins in animal feed and animal-derived food (meat, edible animal tissues, and milk) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In the study, three extraction procedures, as well as various cleanup procedures, were evaluated to select the most suitable sample preparation procedure for different sample matrices. In addition, timed and highly selective reaction monitoring on LC-MS/MS was used to filter out isobaric matrix interferences. The performance characteristics (linearity, sensitivity, recovery, precision, and specificity) of the method were determined according to Commission Decision 2002/657/EC and 401/2006/EC. The established method was successfully applied to screening of mycotoxins in animal feed and animal-derived food. The results indicated that mycotoxin contamination in feed directly influenced the presence of mycotoxin in animal-derived food. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

T-2 and HT-2 (T-2/HT-2) induced cytotoxicity and apoptosis in hepatocytes from broilers. In this study, hepatocytes treated with T-2/HT-2 were analyzed for cytotoxic effects and apoptosis and for the associated mechanisms. To assay cytotoxicity, we used the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) viability assay, hematoxylin-eosin staining and aspartase transaminase and alanine transaminase (ALT/AST) activities. We evaluated apoptosis by fluorescence microscopy using the Terminal transferase nick-end labeling (TUNEL) assay. The apoptotic ratio and the apoptotic stage of the hepatocytes were next assessed with fluorescently labeled (FITC) Annexin V and propidium iodide (PI) staining. Finally, expression levels of apoptosis-related mRNAs were assessed by real-time PCR and those of apoptosis-related proteins by western blotting. We found that cells treated with T-2/HT-2 showed, in a dose dependent manner, significantly lower cell viabilities ( P < 0.05) and markedly increased intercellular spaces, dead cells and ALT/AST activities. T-2/HT-2 treatment also significantly increased the number of apoptotic cells and the apoptotic ratio ( P < 0.05). T-2/HT-2 induced early stage apoptosis of the hepatocytes and levels of apoptosis-related mRNAs and proteins changed in a manner implicating them in the apoptotic process. These changes occurred from 0 to 24 h of T-2/HT-2 exposure. Expression of bax and caspase-7 mRNAs was significantly upregulated, in a time-dependent manner, during this period ( P < 0.05). Levels of mRNAs for caspase-3 and caspase-9 were increased from 0 to 12 h ( P < 0.05) and then decreased after 12 h ( P < 0.05). There were no significant effects on expression of bcl-2 mRNA ( P > 0.05). Expression of all apoptosis-related proteins examined, except for bcl-2, was significantly increased from 0 to 24 h in a time-dependent manner ( P < 0.05). Overall, T-2/HT-2 induced cytotoxicity and apoptosis in hepatocytes. The resulting changes in mRNA and protein expression were shown that several apoptosis-related proteins were involved in the liver toxicity of these agents. [ABSTRACT FROM AUTHOR]

Abstract Fusarium proliferatum is not only a dangerous plant pathogen but also produces various mycotoxins. The aim of this study was to investigate the influences of different carbon sources on fumonisins (FB 1 and FB 2) production and fungal virulence of F. proliferatum. The use of sucrose as the sole carbon source significantly increased the production of fumonisins in F. proliferatum in comparison to fructose. While F. proliferatum showed the stronger ability to infect banana fruit in the fructose group than the sucrose group, which was further investigated by scanning electron microscope (SEM) observations. Differentially expressed genes (DEGs) between the two groups were revealed by Illumina sequencing, including 1293 up-regulated and 907 down-regulated genes. Among them, many important genes were involved in carbohydrate metabolism, including starch and sucrose, and fructose and mannose metabolism. Additionally, the homologous FUM genes in association with fumonisins biosynthesis, including FUM1 , FUM8 and TUM15 , were up-regulated on sucrose compared with fructose. Furthermore, F. proliferatum cultured on fructose source compared with sucrose source showed higher virulence for colonizing green and mature bananas by up-regulating CWDE (cell wall-degrading enzyme)-related genes, which was further confirmed by qRT-PCR. Hence, the results obtained by RNA-Seq, HPLC-MS/MS and virulence experiments elucidated partially that the use of fructose as the sole carbon source can facilitate the fungal pathogenicity, but depress fumonisins production in F. proliferatum. Graphical abstract Unlabelled Image Highlights • Sucrose increases the fumonisin production in F. proliferatum compared to fructose. • While fructose can enhance its virulence to banana compared to sucrose. • 1293 up-regulated and 907 down-regulated genes were identified between the two groups. • Sucrose increases the expression of FUM genes in F. proliferatum compared to fructose. [ABSTRACT FROM AUTHOR]

The family of zearalenones (ZENs) represents a major group of mycotoxins with estrogenic activity. They are produced by Fusarium fungi and cause adverse effects on human health and animal production. The authors describe here a label-free amperometric immunosensor for the direct determination of ZENs. A glassy carbon electrode (GCE) was first modified with polyethyleneimine-functionalized multi-walled carbon nanotubes. Next, gold and platinum nanoparticles (AuPt-NPs) were electro-deposited. This process strongly increased the surface area for capturing a large amount of antibodies and enhanced the electrochemical performance. In a final step, monoclonal antibody against zearalenone was orientedly immobilized on the electrode, this followed by surface blocking with BSA. The resulting biosensor was applied to the voltammetry determination of ZENs, best at a working voltage of 0.18 V (vs SCE). Under optimized conditions, the method displays a wide linear range that extends from 0.005 to 50 ng mL, with a limit of detection of 1.5 pg mL (at an S/N ratio of 3). The assay is highly reproducible and selective, and therefore provides a sensitive and convenient tool for determination of such mycotoxins. [ABSTRACT FROM AUTHOR]

T-2 and HT-2 toxins belong to mycotoxins which are found in human foods and animal chow. We investigated the toxicity and oxidative stress induced by T-2/HT-2 in broilers and chicken hepatocytes. Maize cultures of Fusarium poae was fed to broilers for 42 d, and the physiological index, biochemical index and expression of mRNAs related to oxidative stress were analyzed. Chicken hepatocytes were treated with different levels of T-2/HT-2, and the following parameters were detected: cell viability, GSH and MDA concentration, LDH leakage, activities of ALT/AST, ROS, GSH-PX, SOD and CAT, and expression of mRNA related to oxidative stress. In vivo, high levels of mycotoxins (4 mg/kg T-2 and 0.667 mg/kg HT-2) in feed caused significant reductions in body weight, weight gain, and serum total protein, and significant increases in feed conversion ratio, ALP, ALT/AST activities, and expression of mRNA related to oxidative stress. In vitro, cells treated with T-2/HT-2 showed reductions of GSH concentration and significant increases in LDH leakage, ALT/AST ROS, GSH-PX, SOD and CAT activities, MDA concentration, and expression of mRNA related to oxidative stress. Consequently, F. poae culture material and T-2/HT-2 induced toxicity and oxidative stress in vivo and in vitro, respectively. [ABSTRACT FROM AUTHOR]

A reliable liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed for simultaneous determination of nine mycotoxins, i.e., aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1), aflatoxin G2 (AFG2), ochratoxin A (OTA), zearalenone (ZEN), T-2 toxin, HT2 toxin and deoxynivalenol (DON), in fresh fish (muscle and entrails) as well as dried seafoods. Special focus was given to sample pretreatment which is crucial for an accurate and reliable analytical method. With regards to the high complexity of the matrices, extraction solvent, time, and temperature as well as clean-up cartridges were optimized to improve extraction efficiency and reduce matrix effects. The optimum procedure included ultrasound-assisted extraction with acetonitrile/water/acetic acid (79/20/1, v/v/v) at 40 °C for 30 min, defatting with n-hexane and purification by Oasis HLB cartridges. The method was further validated by determining the linearity ( R 2 ≥ 0.9989), sensitivity (limit of detection ≤2 μg/kg, limit of quantitation ≤3 μg/kg), recovery (72.2–119.9%) and precision (≤18.3%) in muscle and entrails of fresh crucian carp ( Carassius carassius ) as well as dried fish products. The method was proven to be suitable for its intended purposes. Mycotoxins of OTA, ZEN and AFB2 have been found in fresh fish and dried seafoods for the first time. [ABSTRACT FROM AUTHOR]