Accurate Identification of Degraded Products of Aflatoxin B1 Under UV Irradiation Based on UPLC-Q-TOF-MS/MS and NMR Analysis

Analysis, purification, and characterization of AFB1 degraded products are vital steps for elucidation of the photocatalytic mechanism. In this report, the UPLC-Q-TOF-MS/MS technique was first coupled with purification and NMR spectral approaches to analyze and characterize degraded products of AFB1 photocatalyzed under UV irradiation. A total of seventeen degraded products were characterized based on the UPLC-Q-TOF-MS/MS analysis, in which seven ones (1–7) including four (stereo) isomers (1,2, 5, and 6) were purified and elucidated by NMR experiments. According to the structural features of AFB1 and degraded products (1–7), the possible photocatalytic mechanisms were suggested. Furthermore, AFB1 and degraded products (1–7) were evaluated against different cell lines. The results indicated that the UPLC-Q-TOF-MS/MS technique combined with purification, NMR spectral experiments, and biological tests was an applicable integrated approach for analysis, characterization, and toxic evaluation of degraded products of AFB1, which could be used to evaluate other mycotoxin degradation processes.

Efficient Degradation of Aflatoxin B1 and Zearalenone by Laccase-like Multicopper Oxidase from Streptomyces thermocarboxydus in the Presence of Mediators

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.

Lumbricus terrestris regulating the ecosystem service/disservice balance in maize (Zea mays) cultivation

Background and aim Plant pathogenic and mycotoxin-producing Fusarium species are globally widespread and lead to large annual yield losses in maize production (ecosystem disservice). Systems with reduced tillage and mulching are particularly under threat. In the present study, the bioregulatory performance (ecosystem service) of the common earthworm species Lumbricus terrestris was analysed regarding the suppression of three economically relevant Fusarium species, and the reduction of their mycotoxins in the maize mulch layer, taking into account the size of maize residues. Methods A mesocosm field experiment was conducted in a reduced tillage long-term field trial on loam soil. Artificially Fusarium-infected maize residues of two size classes were used as a mulch layer. Impacts of the earthworm species on DNA amounts of Fusarium graminearum, F. culmorum, and F. verticillioides and concentrations of the mycotoxins deoxynivalenol (DON), 3-acetyldeoxynivalenol (3-AcDON), and zearalenone (ZEN) were analysed. Results The results reflect that Fusarium regulation by L. terrestris was species-specific and covered the whole spectrum from suppression (F. graminearum) to slight promotion (F. verticillioides). Regarding the mycotoxins, a significant acceleration of the degradation of all three toxins was detected. Fine chopping of the chaff (< 2 cm) did not significantly alter the earthworms’ regulatory capacity. Conclusion While L. terrestris can shift the ecosystem service/disservice balance in both directions with respect to Fusarium regulation, it shifts it towards ecosystem services with respect to mycotoxin degradation. In synergy with adapted agricultural management, this natural bottom-up effect can help to keep soils healthy for sustainable production in the long run.

Mycotoxin degradation by microbial metabolites

Extracellular metabolites of Gliocladium roseum GRZ7 are able to destroy aflatoxin B1 and zearalenone (by 61.9 and 68%, respectively). The determined optimum pH and temperature confirm the enzymatic nature of these metabolites.

Application of laccases for mycotoxin decontamination

Several food commodities can be infected by filamentous fungi, both in the field and during storage. Some of these fungi, under appropriate conditions, are capable of producing a wide range of secondary metabolites, including mycotoxins, which may resist food processing and arise in the final feed and food products. Contamination of these products with mycotoxins still occurs very often and that is why research in this area is valuable and still evolving. The best way to avoid contamination is prevention; however, when it is not possible, remediation is the solution. Enzymatic biodegradation of mycotoxins is a green solution for removal of these compounds that has attracted growing interest over recent years. Due to their ability to detoxify a wide variety of recalcitrant pollutants, laccases have received a lot of attention. Laccases are multi-copper proteins that use molecular oxygen to oxidise various aromatic and non-aromatic compounds, by a radical-catalysed reaction mechanism. Being non-specific, they are capable of degrading a wide range of compounds and the radical species formed can evolve towards both synthetic and degradative processes. The present review provides an overview of structural features, biological functions and catalytic mechanisms of laccases. The utilisation of laccases for mycotoxin degradation is reviewed, as well as shortcomings and future needs related with the use of laccases for mycotoxin decontamination from food and feed.

Qualifying the T-2 Toxin-Degrading Properties of Seven Microbes with Zebrafish Embryo Microinjection Method

T-2 mycotoxin degradation and detoxification efficiency of seven bacterial strains were investigated with zebrafish microinjection method in three steps ((1) determination of mycotoxin toxicity baseline, (2) examination of bacterial metabolites toxicity, (3) identification of degradation products toxicity). Toxicity of T-2 was used as a baseline of toxic effects, bacterial metabolites of strains as control of bacterial toxicity and degradation products of toxin as control of biodegradation were injected into one-cell stage embryos in the same experiment. The results of in vivo tests were checked and supplemented with UHPLC-MS/MS measurement of T-2 concentration of samples. Results showed that the Rhodococcus erythropolis NI1 strain was the only one of the seven tested (R. gordoniae AK38, R. ruber N361, R. coprophilus N774, R. rhodochrous NI2, R. globerulus N58, Gordonia paraffinivorans NZS14), which was appropriated to criteria all aspects (bacterial and degradation metabolites of strains caused lower toxicity effects than T-2, and strains were able to degrade T-2 mycotoxin). Bacterial and degradation metabolites of the NI1 strain caused slight lethal and sublethal effects on zebrafish embryos at 72- and 120-h postinjection. Results demonstrated that the three-step zebrafish microinjection method is well-suited to the determination and classification of different bacterial strains by their mycotoxin degradation and detoxification efficiency.

Effect of thermal processing and traditional flavouring mixture on mycotoxin reduction in pistachio

The effect of heating (roasting and microwave radiation heating) along with a traditional pistachio flavouring mixture (containing verjuice, thyme extract, and sodium chloride) was investigated on reducing aflatoxins (AFs) and ochratoxin A (OTA) in pistachios. The naturally and artificially contaminated samples were soaked in the flavouring mixture (for 5, 10 and 24 h) and then subjected to roasting (at 120 and 150 °C for 50 min) and heating by microwave radiation (6 and 10 min). The residual mycotoxins were determined by high-performance liquid chromatography. The results showed that all treatments were able to reduce mycotoxin content (aflatoxin B1, B2, G1, G2 and OTA) significantly (P<0.05), up to 85.7±2.5% (during roasting) and up to 72.5±2.6% (during heating by microwave radiation). The highest reduction of AFs and OTA (ranging from 51.7±2.3 to 85.7±2.5%) was found when the contaminated (naturally and artificially) samples were soaked in the traditional mixture for 24 h and roasted at 150 °C. It could be concluded that the traditional flavouring method in combination with the roasting process or heating by microwave radiation could be applied as a useful and safe method for mycotoxin degradation in pistachio. Although, complete elimination of mycotoxins was not achieved, the method reduced mycotoxins more than 60% without adverse effect on the taste and appearance of pistachios.

Degradation of Four Major Mycotoxins by Eight Manganese Peroxidases in Presence of a Dicarboxylic Acid

Enzymatic treatment is an attractive method for mycotoxin detoxification, which ideally prefers the use of one or a few enzymes. However, this is challenged by the diverse structures and co-contamination of multiple mycotoxins in food and feed. Lignin-degrading fungi have been discovered to detoxify organics including mycotoxins. Manganese peroxidase (MnP) is a major enzyme responsible for lignin oxidative depolymerization in such fungi. Here, we demonstrate that eight MnPs from different lignocellulose-degrading fungi (five from Irpex lacteus, one from Phanerochaete chrysosporium, one from Ceriporiopsis subvermispora, and another from Nematoloma frowardii) could all degrade four major mycotoxins (aflatoxin B1, AFB1; zearalenone, ZEN; deoxynivalenol, DON; fumonisin B1, FB1) only in the presence of a dicarboxylic acid malonate, in which free radicals play an important role. The I. lacteus and C. subvermispora MnPs behaved similarly in mycotoxins transformation, outperforming the P. chrysosporium and N. frowardii MnPs. The large evolutionary diversity of these MnPs suggests that mycotoxin degradation tends to be a common feature shared by MnPs. MnP can, therefore, serve as a candidate enzyme for the degradation of multiple mycotoxins in food and feed if careful surveillance of the residual toxicity of degradation products is properly carried out.

Development of an UPLC-MS/MS Method for the Analysis of Mycotoxins in Rumen Fluid with and without Maize Silage Emphasizes the Importance of Using Matrix-Matched Calibration

Ruminants are less susceptible to the effects of mycotoxins than monogastric animals as their rumen microbiota are claimed to degrade and/or deactivate at least some of these toxic compounds. However, the mycotoxin degradation is not well-known yet. For this, a sensitive, specific, and accurate analytical method is needed to determine mycotoxins in the rumen fluid. This study aims to develop and thoroughly validate an ultra-performance liquid chromatography tandem mass spectrometry method for the quantitative determination in the rumen fluid of some of the most relevant mycotoxins found in maize silage in Western Europe: deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), mycophenolic acid (MPA), roquefortine C (ROQ-C) and enniatin B (ENN B), as well as their metabolites deepoxy-deoxynivalenol (DOM-1), α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL). As feed is often present in the rumen fluid samples, the potential interaction of feed particles with the mycotoxin extraction and analysis was investigated. Extraction recovery and matrix effects were determined in the rumen fluid with and without maize silage. Differences in those parameters between rumen fluid alone and rumen fluid with maize silage highlight the importance of using matrix-matched calibration curves for the quantification of mycotoxins in rumen fluid samples. A cross-validation of the method with rumen fluid and maize silage demonstrates that this analytical method can be applied in research on rumen fluid samples to investigate the degradation of the reported mycotoxins by rumen microbiota if matrix-matched calibration is performed.