New application of a dye-decolorizing peroxidase immobilized on magnetic nanoparticles for efficient simultaneous degradation of two mycotoxins.

Nowadays the enzymatic approaches are the most promising strategies for mycotoxins detoxification in food stuffs. Herein, the dye-decolorizing peroxidase RhDypB from Rhodococcus jostii was studied for its ability to degrade two mycotoxins in both free and the immobilized enzyme forms. This enzyme was recombinantly expressed and purified, while Fe3O4 nanoparticles were prepared and modified with chitosan as the immobilization carrier. The immobilized enzyme Fe3O4@CS@RhDypB demonstrated degradation rate of 85.61 % toward aflatoxin B1, while it was firstly found to be able to degrade zearalenone with the rate of 86.52 %, at pH 4.0 on 30 °C. The degradation products were identified as aflatoxin Q1 and 15-OH-ZEN respectively. After 5 cycles of reuse, Fe3O4@CS@RhDypB still exhibited degradation rates of 38.50 % and 49.76 % toward the mycotoxins, indicating its high reusability. Moreover, Fe3O4@CS@RhDypB exhibited excellent stability after 10 days of storage. This work identified potential applications of nanoparticle-immobilized enzyme for biodegradation of mycotoxins in food industry.

The anti-aflatoxigenic mechanism of cinnamaldehyde in Aspergillus flavus.

Aflatoxin B1 (AFB1), the predominant and most carcinogenic naturally polyketide, is mainly produced by Aspergillus flavus and Aspergillus parasiticus. Cinnamaldehyde has been reported for inhibiting the growth and aflatoxin biosynthesis in A. flavus. But its molecular mechanism of action still remains largely ambiguous. Here, the anti-aflatoxigenic mechanism of cinnamaldehyde in A. flavus was investigated via a comparative transcriptomic analysis. The results indicated that twenty five of thirty genes in aflatoxin cluster showed down-regulation by cinnamaldehyde although the cluster regulators aflR and aflS were slightly up-regulated. This may be due to the up-regulation of the oxidative stress-related genes srrA, msnA and atfB being caused by the significant down-regulation of the diffusible factor FluG. Cinnamaldehyde also inhibited aflatoxin formation by perturbing GPCRs and oxylipins normal function, cell wall biosynthesis and redox equilibrium. In addition, accumulation of NADPH due to up-regulation of pentose phosphate pathway drove acetyl-CoA to lipids synthesis rather than polyketides. Both GO and KEGG analysis suggested that pyruvate and phenylalanine metabolism, post-transcriptional modification and key enzymes biosynthesis might be involved in the suppression of AFB1 production by cinnamaldehyde. This study served to decipher the anti-aflatoxigenic properties of cinnamaldehyde in A. flavus and provided powerful evidence for its use in practice.

Interaction of water activity and temperature on the growth, gene expression and aflatoxin production by Aspergillus flavus on paddy and polished rice.

Water activity (aw) and temperature are two pivotal environmental factors affecting Aspergillus flavus growth and aflatoxin production. Here, we found that AFB1 production on polished rice can occur over a wider range of temperature × aw levels than that on paddies. For fungal growth on polished rice, the optimum conditions were aw 0.92-0.96 and 28-37 °C. The maximum amounts of AFB1 on polished rice was observed at 33 °C and aw 0.96. Compared to 33 °C, all tested genes of A. flavus on polished rice were significantly up-regulated at 25 °C under aw 0.96. The late structural genes of pathway were significantly down-regulated at 37 °C under aw 0.96, although aflR and aflS and most of early structural genes were up-regulated. Compared to aw 0.96, most of pathway genes were significantly down-regulated at aw 0.90 and 0.99 under 33 °C, although two regulatory genes were up-regulated at aw 0.90.

Ethanol Inhibits Aflatoxin B1 Biosynthesis in Aspergillus flavus by Up-Regulating Oxidative Stress-Related Genes.

As the most carcinogenic, toxic, and economically costly mycotoxins, aflatoxin B1 (AFB1) is primarily biosynthesized by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin biosynthesis is related to oxidative stress and functions as a second line of defense from excessive reactive oxygen species. Here, we find that ethanol can inhibit fungal growth and AFB1 production by A. flavus in a dose-dependent manner. Then, the ethanol's molecular mechanism of action on AFB1 biosynthesis was revealed using a comparative transcriptomic analysis. RNA-Seq data indicated that all the genes except for aflC in the aflatoxin gene cluster were down-regulated by 3.5% ethanol. The drastic repression of aflatoxin structural genes including the complete inhibition of aflK and aflLa may be correlated with the down-regulation of the transcription regulator genes aflR and aflS in the cluster. This may be due to the repression of several global regulator genes and the subsequent overexpression of some oxidative stress-related genes. The suppression of several key aflatoxin genes including aflR, aflD, aflM, and aflP may also be associated with the decreased expression of the global regulator gene veA. In particular, ethanol exposure caused the decreased expression of stress response transcription factor srrA and the overexpression of bZIP transcription factor ap-1, C2H2 transcription factors msnA and mtfA, together with the enhanced levels of anti-oxidant enzymatic genes including Cat, Cat1, Cat2, CatA, and Cu, Zn superoxide dismutase gene sod1. Taken together, these RNA-Seq data strongly suggest that ethanol inhibits AFB1 biosynthesis by A. flavus via enhancing fungal oxidative stress response. In conclusion, this study served to reveal the anti-aflatoxigenic mechanisms of ethanol in A. flavus and to provide solid evidence for its use in controlling AFB1 contamination.

Large-Scale Comparative Analysis of Eugenol-Induced/Repressed Genes Expression in Aspergillus flavus Using RNA-seq.

Aflatoxin B1 (AFB1), which is mainly produced by Aspergillus flavus and Aspergillus parasiticus, is the most toxic and hepatocarcinogenic polyketide known. Chemical fungicides are currently utilized to reduce this fungal contaminant, but they are potentially harmful to human health and the environment. Therefore, natural anti-aflatoxigenic products are used as sustainable alternatives to control food and feed contamination. For example, eugenol, presents in many essential oils, has been identified as an aflatoxin inhibitor. However, its exact mechanism of inhibition is yet to be clarified. In this study, the anti-aflatoxigenic mechanism of eugenol in A. flavus was determined using a comparative transcriptomic approach. Twenty of twenty-nine genes in the aflatoxin biosynthetic pathway were down-regulated by eugenol. The most strongly down-regulated gene was aflMa, followed by aflI, aflJ, aflCa, aflH, aflNa, aflE, aflG, aflM, aflD, and aflP. However, the expression of the regulator gene aflR did not change significantly and the expression of aflS was slightly up-regulated. The down-regulation of the global regulator gene veA resulted in the up-regulation of srrA, and the down-regulation of ap-1 and mtfA. The early developmental regulator brlA was profoundly up-regulated in A. flavus after eugenol treatment. These results suggested a model in which eugenol improves fungal development by up-regulating the expression of brlA by the suppression of veA expression and inhibits aflatoxin production through the suppression of veA expression. Exposure to eugenol also caused dysregulated transcript levels of the G protein-coupled receptors (GPCRs) and oxylipins genes. A Gene Ontology analysis indicated that the genes that were highly responsive to eugenol were mainly enriched in RNA-binding functions, suggesting that post-transcriptional modification plays a pivotal role in aflatoxin biosynthesis. KEGG analysis showed that ribosome biogenesis was the most dysregulated pathway, suggesting that eugenol dysregulates ribosome biogenesis, which then interrupts the biosynthesis of Nor-1, Ver-1, and OmtA, and prevents aflatoxisomes performing their normal function in aflatoxin production. In conclusion, our results indicated that eugenol inhibited AFB1 production by modulating the expression of structural genes in aflatoxin pathway, fungal antioxidant status, post-transcriptional modifications and biosynthesis of backbone enzymes in A. flavus.