Influence of Two Garlic-Derived Compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane Thiosulfinate (PTSO), on Growth and Mycotoxin Production by Fusarium Species In Vitro and in Stored Cereals.

Two garlic-derived compounds, Propyl Propane Thiosulfonate (PTS) and Propyl Propane Thiosulfinate (PTSO), were examined for their efficacy against mycotoxigenic Fusarium species (F. graminearum, F. langsethiae, F. verticillioides). The objectives were to assess the inhibitory effect of these compounds on growth and mycotoxin production in vitro, and in situ in artificially inoculated wheat, oats and maize with one isolate of each respectively, at different water activity (aw) conditions when stored for up to 20 days at 25 °C. In vitro, 200 ppm of either PTS or PTSO reduced fungal growth by 50-100% and mycotoxin production by >90% depending on species, mycotoxin and aw conditions on milled wheat, oats and maize respectively. PTS was generally more effective than PTSO. Deoxynivalenol (DON) and zearalenone (ZEN) were decreased by 50% with 80 ppm PTSO. One-hundred ppm of PTS reduced DON and ZEN production in wheat stored at 0.93 aw for 20 days, although contamination was still above the legislative limits. Contrasting effects on T-2/HT-2 toxin contamination of oats was found depending on aw, with PTS stimulating production under marginal conditions (0.93 aw), but at 0.95 aw effective control was achieved with 100 ppm. Treatment of stored maize inoculated with F. verticilliodies resulted in a stimulation of total fumonsins in most treatments. The potential use of such compounds for mycotoxin control in stored commodities is discussed.

T-2 Toxin/HT-2 Toxin and Ochratoxin A ELISAs Development and In-House Validation in Food in Accordance with the Commission Regulation (EU) No 519/2014.

T-2 toxin/HT-2 toxin (T-2/HT-2) and ochratoxin A (OTA) are mycotoxins that can contaminate a variety of agricultural commodities. To protect consumers' health, indicative limits for T-2/HT-2 and maximum limits for OTA have been set by the European Commission, requiring food business operators and controlling agencies to conduct routine checks for the presence of these harmful contaminants. Screening methods are increasingly used for monitoring purposes. Due to the demand for new and improved screening tools, two individual detection methods, T-2/HT-2 and OTA enzyme-linked immunosorbent assays (ELISAs), were developed in this study. The T-2/HT-2 ELISA was based on a T-2 monoclonal antibody with an IC50 (50% inhibitory concentration) of 0.28 ng/mL and 125% cross-reactivity with HT-2. As regards the OTA ELISA, a new sensitive monoclonal antibody specific to OTA with an IC50 of 0.13 ng/mL was produced. Both developed ELISA tests were then validated in agricultural commodities in accordance with the new performance criteria guidelines for the validation of screening methods for mycotoxins included in Commission Regulation (EU) No 519/2014. The T-2/HT-2 ELISA was demonstrated to be suitable for the detection of T-2/HT-2 in cereals and baby food at and above the screening target concentration (STC) of 12.5 µg/kg and 7.5 µg/kg, respectively. The OTA ELISA was shown to be applicable for the detection of OTA in cereals, coffee, cocoa and wine at and above the STC of 2 µg/kg, 2.5 µg/kg, 2.5 µg/kg and 0.4 ng/mL, respectively. The accuracy of both ELISAs was further confirmed by analysing proficiency test and reference samples. The developed methods can be used for sensitive and high-throughput screening for the presence of T-2/HT-2 and OTA in agricultural commodities.

Selenium promotes metabolic conversion of T-2 toxin to HT-2 toxin in cultured human chondrocytes.

To explore the metabolism of T-2 toxin in human chondrocytes (HCs) and determine the impact of selenium supplementation. For determination of cytotoxicity using the MTT assay, optical density values were read with an automatic enzyme-linked immunosorbent assay reader at 510nm. Cell survival was calculated and the cytotoxicity estimated. To identify the metabolites of T-2 toxin, the medium supernatants and C28/I2 cells were analyzed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) separately. For HPLC-MS/MS, the mobile phase A was water and phase B was 98% methanol. The gradient for the elution was: 0-0.5min, 50% of B; 0.5-2.0min, 100% of B; 2.0-3.5min, 100% of B; 3.6-6min, 50% of B. T-2 toxin increased the toxicity to C28/I2 cells significantly in a dose- and time-dependent manner (viability range 91.5-22.0%). Supplementation with selenium (100ng/mL) could increase the cell viability after the 24h incubation. The concentration of T-2 toxin in the cell medium decreased from 20 to 6.67±1.02ng/mL, and the concentration of HT-2 toxin increased from 0 to 6.88±1.23ng/mL during the 48h incubation, whereas the relative concentration of T-2 toxin in cells increased from 0 to 12.80±1.84ng/g. Supplementary selenium in the HCs cultures reduced the cytotoxicity induced by T-2 toxin significantly, and was associated with rapid conversion of T-2 toxin in the culture medium to HT-2 toxin. T-2 toxin was more toxic to HCs than HT-2 toxin at equivalent concentrations. HT-2 toxin was a detectable metabolite of T-2 toxin in cultured HCs, and selenium enhanced the metabolic conversion of T-2 toxin, reducing its cytotoxicity to HCs.

Determination of T-2 and HT-2 Toxins in Oats and Oat-Based Breakfast Cereals by Liquid-Chromatography Tandem Mass Spectrometry.

This protocol specifies an accurate and sensitive method for the determination of T-2 and HT-2 toxins content in oats and oat-based foods using liquid chromatography with tandem mass spectrometry detection. T-2 and HT-2 toxins are extracted from the test material with a mixture of acetonitrile and water. The filtered extract is dried, reconstituted with a mixture of methanol and water, then purified on a polymeric solid-phase extraction cartridge. Toxins are finally eluted from the column with methanol and quantified by reversed phase high performance liquid chromatography with tandem mass spectrometry detection.

Iron (II, III) oxide/multi-walled carbon nanotube composite as solid-phase extraction sorbent followed by ultra-high performance liquid chromatography tandem mass spectrometry for simultaneous determination of zearalenone and type A trichothecenes in Salviae miltiorrhizae Radix et Rhizoma (Danshen).

For the first time, a reliable solid-phase extraction (SPE) procedure using iron (II, III) oxide (Fe3O4)/multi-walled carbon nanotube (MWCNT) composite as sorbents was proposed for purification and enrichment of zearalenone (ZEA) and four type A trichothecenes (T-2 toxin (T-2), HT-2 toxin (HT-2), neosolaniol (NEO) and diacetoxyscirpenol (DAS)) in Salviae miltiorrhiza Radix et Rhizoma (Danshen). The Fe3O4/MWCNT composite was synthesized by assembling Fe3O4 with MWCNT by sonication through an "aggregation wrap" mechanism and several key parameters affecting the performance of SPE procedure such as the composition of sample loading solutions, washing and elution solvents were thoroughly investigated. After optimization, 2% acetonitrile aqueous solution as the loading solution, 5% methanol aqueous solution as the washing solution and acetone containing 0.5% formic acid as the elution solvent presented an excellent purification efficiency for the five targets in Danshen. Under the optimal sample pretreatment conditions followed by analysis with ultra-high performance liquid chromatography-tandem mass spectrometry, satisfactory linearity (R2≥0.991), high sensitivity (limit of quantification in the range of 1.20-4.80µgkg-1), good recovery (73.7-91.9%) and acceptable precision (RSD, 2.1-13.3%) were obtained. The applicability of the validated method was further verified in real Salviae miltiorrhiza Radix et Rhizoma samples.

Toxicity induced by F. poae-contaminated feed and the protective effect of Montmorillonite supplementation in broilers.

The T-2 and HT-2 toxins, the main metabolites of Fusarium poae, induce toxicity in broilers and accumulate in tissues. Consequently, during the breeding process of broilers, diets are frequently supplemented with physical adsorbents to protect birds against the toxicity induced by mycotoxins. In the present research, T-2 and HT-2 were produced in maize inoculated with F. poae. Mont, the strongest adsorbent based on in vitro adsorption ratios, was added to the contaminated diet. One-day-old chickens were randomly and equally divided into the following four groups: control diet group, Mont supplemented diet group, contaminated diet group and detoxification diet group. The experiment lasted for 42 days. Compared to the control group, the contaminated group showed significant decrease in body weight, feed intake and TP (P < 0.05), and marked increase in FCR, ALP, AST and ALT activity, T-2/HT-2 residues in the tissues and the relative expressions of apoptosis-related mRNAs (P < 0.05). Mont supplementation provided protection for the treated broilers in terms of performance, blood biochemistry, hepatic function, T-2/HT-2 residue of tissues and apoptosis. Therefore, Mont may be suitable as a detoxification agent for T-2/HT-2 in feed for broilers.

Validation of a gas chromatography-electron capture detection of T-2 and HT-2 toxins in Chinese herbal medicines and related products after immunoaffinity column clean-up and pre-column derivatization.

A sensitive, reproducible and accurate gas chromatography-electron capture detection (GC-ECD) method was developed for simultaneous determination of T-2 and HT-2 toxins in Chinese herbal medicines (CHMs) and related products after immunoaffinity column (IAC) clean-up and pre-column derivatization with N-heptafluoro-butyryl imidazole (HFBI). Then, gas chromatography-spectrometry spectrometer (GC-MS) was applied to confirm the positive results and interfering peaks. The limits of detection (LODs) for T-2 and HT-2 toxins were 1.88 and 0.47ng/g, and the recoveries for different CHMs ranged from 89.2% to 99.1% with relative standard deviation (RSD) <6.0% for T-2 and from 85.9% to 99.0% with RSD <8.8% for HT-2 toxin, respectively. The validated method was successfully applied for the determination of T-2 and HT-2 toxins in 89 Chinese herbal medicines and 10 related products from various sources, where it was found that T-2 and HT-2 toxins were not detected in any of the tested samples. These results were reliable by confirmation using GC-MS. Some unknown peaks were interfering peaks not the target toxins.