Mycotoxin risk management in maize gluten meal.

Maize gluten meal (MGM) is a by-product of maize starch and ethanol, produced by the wet milling process. Its high protein content makes it a preferred ingredient in feed. Given the high prevalence of mycotoxins in maize globally, they pose a significant challenge to use of MGM for feed: wet milling could concentrate certain mycotoxins in gluten components, and mycotoxin consumption affects animal health and can contaminate animal-source foods. To help confront this issue, this paper summarizes mycotoxin occurrence in maize, distribution during MGM production and mycotoxin risk management strategies for MGM through a comprehensive literature review. Available data emphasize the importance of mycotoxin control in MGM and the necessity of a systematic control approach, which includes: good agriculture practices (GAP) in the context of climate change, degradation of mycotoxin during MGM processing with SO2 and lactic acid bacteria (LAB) and the prospect of removing or detoxifying mycotoxins using emerging technologies. In the absence of mycotoxin contamination, MGM represents a safe and economically critical component of global animal feed. With a holistic risk assessment-based, seed-to-MGM-feed systematic approach to reducing and decontaminating mycotoxins in maize, costs and negative health impacts associated with MGM use in feed can be effectively reduced.

Development of a Linear Immobilization Carrier-Based Immunoassay for Aflatoxin.

We explored the feasibility of developing immunoassay technology with a linear carrier, to develop a simpler and cheaper rapid immunoassay technology. We selected aflatoxins as an example for research, as they are a group of highly toxic and carcinogenic compounds representing a worldwide threat to human health and life. With a non-competitive immunoassay, we detected and evaluated the effect of 28 different linear materials on antibody immobilization. Mercerized cotton and Dyneema line were chosen from the linear materials for further comparison using a competitive immunoassay, because both showed high-signal values and relatively low background noise. The results showed the sensitive IC50 of mercerized cotton as the reaction carrier was 0.33 ng/mL, and the linear range was 0.16~3.25 ng/mL. The sensitivity using Dyneema line as the reaction carrier was 1.16 ng/mL. The competitive curves of four sample matrices were established to evaluate the stability of the detection system; these were basically consistent with those without sample matrices. In conclusion, both mercerized cotton and Dyneema, will be suggested for the novel development of linear immobilization carrier-based immunoassays for other analytes, and especially to construct inexpensive and easy-to-obtain biological and environmental analytical technologies and biosensors.

A Facile Route to Synthesis of Hierarchically Porous Carbon via Micelle System for Bifunctional Electrochemical Application.

Well-ordered hierarchically porous carbon (HPC) nanomaterials have been successfully synthesized by a facile, efficient, and fast heated-evaporation induced self-assembly (HISA) method. A micelle system was employed as the template by using the HISA method for the first time, which possessed great potential in the large-scale production of HPC materials. Various surfactants, including triblock copolymer Pluronic F127, P123, F108, and cationic CTAB, were used in the polymerization process as templates to reveal the relationship between the structure of surfactants and architecture of the as-prepared HPCs. Transmission electron microscopy (TEM), X-ray diffraction (XRD), Nitrogen adsorption, and Fourier transform infrared (FTIR) measurements were conducted to investigate the morphology, structure, and components of HPCs, which further confirmed the well-ordered and uniform mesoporous structure. The as-prepared HPC sample with F127 possessed the largest specific surface area, suitable pore size, and well-ordered mesoporous structure, resulting in better electrochemical performance as electrodes in the fields of energy storage and conversion system. Doped with the metallic oxide MnO2, the MnO2/HPC composites presented the outstanding electrochemical activity in supercapacitor with a high specific capacitance of 531.2 F g-1 at 1 A g-1 and excellent cycling performance with little capacity fading, even after 5,000 cycles. Moreover, the obtained sample could also be applied in the fields of oxygen reduction reaction (ORR) for its abundant active sites and regulate architecture. This versatile approach makes the mass industrial production of HPC materials possible in electrochemical applications through a facile and fast route.

Inhibitory effect of allyl and benzyl isothiocyanates on ochratoxin a producing fungi in grape and maize.

The purpose of this study was to evaluate the inhibitory effect of allyl-isothiocyanate (AITC) and benzyl-isothiocyanate (BITC) on fungal growth and Ochratoxin A (OTA) production by Aspergillus ochraceus, A. carbonarius and A. niger. Here, we found that spore germination and fungal growth of the three fungi were significantly inhibited when the concentration of AITC and BITC was higher than 1.25 µg/mL. The inhibitory effect of AITC or BITC on A. carbonaceus and A. ochraceus was significantly stronger than that of A. niger. Scanning electron microscopy showed that the mycelia of all three fungi were changed by AITC and BITC. Compared with A. ochraceus and A. carbonarius, the damage to A. niger was lower. For OTA production, AITC and BITC could significantly down-regulated the expression of all five OTA biosynthesis genes in A. niger and A. carbonarius. In A. ochraceus, although several OTA biosynthesis genes were up-regulated, the key PKS gene was down-regulated by AITC and BITC. Twenty-five µg/mL of AITC or BITC could reduce the infection of the three fungi on grapes with inhibition rates of 28%-36% during 14 days and prolong the shelf life of grapes. In maize, the OTA production of the three fungi was significantly reduced by 25 µg/mL of AITC and BITC with the inhibition rates 68.04%-93.49% and 65.87%-75.45%, respectively. These results suggest that AITC and BITC can be used as natural fungicides to prevent A. niger, A. carbonarius and A. ochraceus from infecting grapes and maize and control OTA contamination.

[High expression of zearalenone degrading enzyme in Pichia pastoris].

High expression of zearalenone (ZEN) degrading enzyme gene (zlhy-6) in Pichia pastoris strain GS115 was achieved by codon optimization and multi-copy construction in vitro. The codon-optimized zlhy-6 gene sequence was synthesized with the alpha factor signal peptide coding sequence and inserted into the pAO815 plasmid. The expression plasmid containing 1-6 expression cassettes was constructed by enzyme digestion and transferred into P. pastoris GS115 strain to obtain the ZEN degrading enzyme recombinant strain. The molecular weight of the recombinant protein was 28.9 kDa, which was consistent with the theoretical value. After 3 days of induction fermentation, the protein concentration reached the highest level and then decreased; the expression level was the highest in the induction culture at pH 5.0 and 4.5, while the expression level at other pH was very low; the expression level was the highest when 0.8% methanol was added every day and 10% inoculation was added; the expression level of four-copy transformants was the highest, and the enzyme activity reached 10 U/mL after 3 days of flask fermentation, The degradation rate of ZEN in 1 g corn ballast was 44.08%-75.51% when 0.1-0.5 mL fermentation supernatant added and hydrolyzed for 24 hours. The results of this study laid a foundation for improving the industrial fermentation level of ZEN degrading enzyme and its application in eliminating ZEN in food and feed.

El factor de transcripción bZIP Afap1 afecta al estrés oxidativo y la biosíntesis de aflatoxinas en Aspergillus flavus / The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus

Abstract Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap 1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H 2O 2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H 2O 2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB 1 in the mutant strains was approximately V of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus. ©2018 Published by Elsevier España, S.L.U. on behalf of Asociación Argentina de Microbiología. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/ licenses/by-nc-nd/4.0/).

Resumen La aflatoxina es un metabolito secundario cancerígeno producido principalmente por Aspergillus flavus y Aspergillus parasiticus, que pone en riesgo grave a la salud de los humanos y los animales. El estrés oxidativo es una respuesta de defensa común, y es sabido que las especies reactivas de oxígeno (ROS) pueden inducir la síntesis de una serie de metabolitos secundarios, incluida la aflatoxina. Empleando mutantes carentes del gen afap1 se evaluó el papel de Afap1 en el estrés oxidativo y la síntesis de aflatoxinas. El crecimiento de las cepas mutadas se vio significativamente inhibido con el aumento de la concentración de H 2O 2, la inhibición fue completa a 40mmol/l. Sin embargo, en el análisis cuantitativo por HPLC, la concentración de la aflatoxina AFBi aumentó con el aumento de la concentración de H 2O 2 hasta 10mmol/l. Tras un análisis apoyado en la información provista por la herramienta NCBI BLAST, se supuso que Afap1, un factor de transcripción de la cremallera de leucina básica (bZIP), estaba asociado con el estrés oxidativo en este hongo. El tratamiento con 5mmol/l de H 2O 2 inhibió completamente el crecimiento de las cepas mutantes en afap1, pero no afectó el crecimiento de la cepa CA14PTs (cepa no mutada). Además, la concentración de AFB 1 en las cepas mutadas fue de aproximadamente 1/4 de la observada en CA14PTs. Estos resultados sugieren que Afap1 juega un papel clave en la regulación del estrés oxidativo y la producción de aflatoxinas en A. flavus.

Morphological and Transcriptomic Analysis of the Inhibitory Effects of Lactobacillus plantarum on Aspergillus flavus Growth and Aflatoxin Production.

Lactobacillus plantarum, as a natural bio-preservative, has attracted a great deal of attention in recent years. In this study, 22 L. plantarum strains were tested against the aflatoxin-producing fungus, Aspergillus flavus; strain IAMU80070 showed the highest antifungal activity. At a concentration of 5 × 105 colony-forming units (CFU) mL-1, it completely inhibited A. flavus growth and decreased aflatoxin production by 93%. Furthermore, ultrastructural examination showed that IAMU80070 destroyed the cellular structure of hyphae and spores. To explore the inhibitory effect of IAMU80070 on A. flavus at the transcriptional level, transcriptome data were obtained and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The aflatoxin biosynthetic process was the most significantly downregulated functional category, while genes implicated in the synthesis and organization of cell wall polysaccharides were upregulated. Quantitative real-time PCR results verified the credibility and reliability of the RNA sequencing data. These results provided insight into the transcriptome of A. flavus in response to the antagonistic effects of L. plantarum IAMU80070.

The Complex Essential Oils Highly Control the Toxigenic Fungal Microbiome and Major Mycotoxins During Storage of Maize.

The contamination of maize with fungi and subsequent mycotoxins is a pivotal and long-standing safety concern in the maize industry. In this study, the inhibitory effects of the complex essential oils (cinnamaldehyde, citral, eugenol, and menthol, 3:3:2:2, v/v) on fungal growth and mycotoxins production in stored maize were evaluated using traditional plate counting, internal transcribed spacer 2 (ITS2) sequencing and liquid chromatography-tandem mass spectrometry. Complex essential oils (0.02%) significantly (p < 0.05) reduced the total fungi counts and the content of aflatoxin B1, zearalenone, and deoxynivalenol in stored maize during 12 months of storage, and were more effective than propionic acid (0.2%). The fungal diversity of the control group was the highest with 113 operational taxonomic units. During storage of maize kernels, Aspergillus, Fusarium, Wallemia, Sarocladium, and Penicillium were main genera. At 0-6 months, the fungal diversity was high and Fusarium was predominant genus. However, at 7-11 months, the fungal diversity was low and Aspergillus was predominant genus. During the later stages of storage, the prevalence of Aspergillus in maize treated with essential oils was significantly lower than (p < 0.05) that observed in the propionic acid treated and control samples. The results of this study suggest that the complex essential oils may be employed successfully to control toxigenic fungi and subsequent contamination with mycotoxins in maize.

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.

The bZIP transcription factor Afap1 mediates the oxidative stress response and aflatoxin biosynthesis in Aspergillus flavus.

Aflatoxin is a carcinogenic secondary metabolite produced mainly by Aspergillus flavus and Aspergillus parasiticus, which can seriously endanger the health of humans and animals. Oxidative stress is a common defense response, and it is known that reactive oxygen species (ROS) can induce the synthesis of a series of secondary metabolites, including aflatoxin. By using mutants lacking the afap 1 gene, the role of afap1 gene in oxidative stress and aflatoxin synthesis was assessed. The growth of the mutant strains was significantly inhibited by the increase in the concentration of H2O2, inhibition was complete at 40mmol/l. However, in the quantitative analysis by HPLC, the concentration of AFB1 increased with the increased H2O2 until 10mmol/l. Following an analysis based on the information provided by the NCBI BLAST analysis, it was assumed that Afap1, a basic leucine zipper (bZIP) transcription factor, was associated with the oxidative stress in this fungus. Treatment with 5mmol/l H2O2 completely inhibited the growth of the mutant strains in afap 1 but did not affect the growth of the CA14PTs strain (non-mutant strain). In addition, the concentration of AFB1 in the mutant strains was approximately » of that observed in the CA14PTs strain. These results suggested that Afap1 plays a key role in the regulation of oxidative stress and aflatoxin production in A. flavus.

Deoxynivalenol in wheat from the Northwestern region in China.

Because of global warming and changes in farming systems, Fusarium head blight has gradually spread towards high-latitude regions such as Northwestern China. A survey was conducted to assess the prevalence and concentration of deoxynivalenol (DON) in wheat harvested during 2013 from the Shaanxi, Ningxia, Gansu, and Xinjiang provinces in China. DON concentration in 181 wheat samples was analysed by high-performance liquid chromatography combined with ultraviolet detection. Of the wheat samples, 82.9% were contaminated with DON, with a mean DON concentration of 500 µg/kg. According to the Chinese standard limits for DON, 10% of the positive samples were above the maximum limit of 1000 µg/kg. Regions with higher humidity showed higher levels of DON in the wheat samples. These results show the necessity of raising awareness of DON contamination in people from Northwestern China to protect their health from the risk of exposure to DON.

Effect of Cinnamaldehyde on Morphological Alterations of Aspergillus ochraceus and Expression of Key Genes Involved in Ochratoxin A Biosynthesis.

Ochratoxin A (OTA) is a potent nephrotoxic, hepatotoxic, and teratogenic compound which is a significant mycotoxin contaminates cereals during storage. Aspergillus ochraceus is the most common producer of OTA in cereals and cereal-derived products. Cinnamaldehyde is a natural substance derived from plant cinnamon playing an important role in the reduction of OTA contamination. In this study, the antifungal and antitoxigenic effect of cinnamaldehyde was investigated with its mechanisms of inhibition of fungal growth at the morphological and ultrastructural levels, and inhibition of OTA biosynthesis at the transcriptional level. Significant A. ochraceus growth was inhibited at 0.4⁻1.6 mmol/L with fumigation. A. ochraceus exposed to 0.4 mmol/L of cinnamaldehyde indicated irreversible harmful morphological and ultrastructural modifications such as the folding of the cell, the loss of integrity of the cell wall, the disruption of plasma membrane, the destruction of the mitochondria, and the absence of intracellular organelles. These alterations may be attributed to its inhibition of enzymatic reactions that regulate cell wall synthesis, thus disturbing the morphogenesis and growth of A. ochraceus. In the presence of cinnamaldehyde, the tested biosynthetic and regulatory genes like pks, nrps, veA, laeA and velB were highly downregulated. Moreover, the downregulation effect of cinnamaldehyde increased proportionally with the concentrations. These results suggest that the decrease of OTA production by cinnamaldehyde is attributed to the downregulation of the transcriptional levels of OTA biosynthetic and regulatory genes besides the inhibition of fungal growth. The study reveals the mechanisms of the antifungal and antitoxigenic activities of cinnamaldehyde against A. ochraceus, and further emphasizes that cinnamaldehyde could be a safe and effective natural agents against OTA contamination during cereals storage.

A Consensus Ochratoxin A Biosynthetic Pathway: Insights from the Genome Sequence of Aspergillus ochraceus and a Comparative Genomic Analysis.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by Aspergillus and Penicillium species that widely contaminates food and feed. We sequenced and assembled the complete ∼37-Mb genome of Aspergillusochraceus fc-1, a well-known producer of OTA. Key genes of the OTA biosynthetic pathway were identified by comparative genomic analyses with five other sequenced OTA-producing fungi: A. carbonarius, A. niger, A. steynii, A. westerdijkiae, and Penicillium nordicum OTA production was completely inhibited in the deletion mutants (ΔotaA, ΔotaB, ΔotaC, ΔotaD, and ΔotaR1), and OTA biosynthesis was restored by feeding a postblock substrate to the corresponding mutant. The OTA biosynthetic pathway was unblocked in the ΔotaD mutant by the addition of heterologously expressed halogenase. OTA biosynthesis begins with a polyketide synthase (PKS), OtaA, utilizing acetyl coenzyme A (acetyl-CoA) and malonyl-CoA to synthesize 7-methylmellein, which is oxidized to OTß by cytochrome P450 monooxygenase (OtaC). OTß and l-ß-phenylalanine are combined by a nonribosomal peptide synthetase (NRPS), OtaB, to form an amide bond to synthesize OTB. Finally, OTB is chlorinated by a halogenase (OtaD) to OTA. The otaABCD genes were expressed at low levels in the ΔotaR1 mutant. A second regulator, otaR2, which is adjacent to the biosynthetic gene, could modulate only the expression of otaA, otaB, and otaD Thus, we have identified a consensus OTA biosynthetic pathway that can be used to prevent and control OTA synthesis and will help us understand the variation and production of the intermediate components in the biosynthetic pathway.IMPORTANCE Ochratoxin A (OTA) is a significant mycotoxin that contaminates cereal products, coffee, grapes, wine, cheese, and meat. OTA is nephrotoxic, carcinogenic, teratogenic, and immunotoxic. OTA contamination is a serious threat to food safety, endangers human health, and can cause huge economic losses. At present, >20 species of the genera Aspergillus and Penicillium are known to produce OTA. Here we demonstrate that a consensus OTA biosynthetic pathway exists in all OTA-producing fungi and is encoded by a gene cluster containing four highly conserved biosynthetic genes and a bZIP transcription factor.

pH-Signaling Transcription Factor AopacC Regulates Ochratoxin A Biosynthesis in Aspergillus ochraceus.

In Aspergillus and Penicillium species, an essential pH-response transcription factor pacC is involved in growth, pathogenicity, and toxigenicity. To investigate the connection between ochratoxin A (OTA) biosynthesis and ambient pH, the AopacC in Aspergillus ochraceus was functionally characterized using a loss-of-function mutant. The mycelium growth was inhibited under pH 4.5 and 10.0, while the sporulation increased under alkaline condition. A reduction of mycelium growth and an elevation of sporulation was observed in Δ AopacC mutant. Compared to neutral condition, OTA contents were respectively reduced by 71.6 and 79.8% under acidic and alkaline conditions. The expression of AopacC increased with the elevated pH, and deleting AopacC dramatically decreased OTA production and biosynthetic genes Aopks expression. Additionally, the Δ AopacC mutant exhibited attenuated infection ability toward pear fruits. These results suggest that AopacC is an alkaline-induced regulator responsible for growth and OTA biosynthesis in A. ochraceus and this regulatory mechanism might be pH-dependent.

The Mycotoxin Zearalenone Hinders Candida albicans Biofilm Formation and Hyphal Morphogenesis.

Yeast-mold mycobiota inhabit several natural ecosystems, in which symbiotic relationships drive strategic pathoadaptation. Mycotoxins are metabolites produced by diverse mycotoxigenic fungi as a defense against yeasts, though at times yeasts secrete enzymes that degrade, detoxify, or bio-transform mycotoxins. The present study is focused on the in vitro inhibitory effects of zearalenone (ZEN), a F2 mycotoxin produced by several Fusarium and Gibberella species, on different microbial strains. ZEN exhibited no effect on the planktonic growth or biofilms of several Gram positive and negative bacteria at the tested concentrations. Remarkably, Candida albicans biofilm formation and hyphal morphogenesis were significantly inhibited when treated with 100 µg/mL of ZEN. Likewise, ZEN proficiently disrupted pre-formed C. albicans biofilms without disturbing planktonic cells. Furthermore, these inhibitions were confirmed by crystal violet staining and XTT reduction assays and by confocal and scanning electron microscopy. In an in vivo model, ZEN significantly suppressed C. albicans infection in the nematode Caenorhabditis elegans. The study reports the in vitro antibiofilm efficacy of ZEN against C. albicans strains, and suggests mycotoxigenic fungi participate in asymmetric competitive interactions, such as, amensalism or antibiosis, rather than commensal interactions with C. albicans, whereby mycotoxins secreted by fungi destroy C. albicans biofilms.

Aflatoxin B1 inhibition in Aspergillus flavus by Aspergillus niger through down-regulating expression of major biosynthetic genes and AFB1 degradation by atoxigenic A. flavus.

Twenty Aspergillus niger strains were isolated from peanuts and 14 strains were able to completely inhibit AFB1 production with co-cultivation. By using a Spin-X centrifuge system, it was confirmed that there are some soluble signal molecules or antibiotics involved in the inhibition by A. niger, although they are absent during the initial 24h of A. flavus growth when it is sensitive to inhibition. In A. flavus, 19 of 20 aflatoxin biosynthetic genes were down-regulated by A. niger. Importantly, the expression of aflS was significantly down-regulated, resulting in a reduction of AflS/AflR ratio. The results suggest that A. niger could directly inhibit AFB1 biosynthesis through reducing the abundance of aflS to aflR mRNAs. Interestingly, atoxigenic A. flavus JZ2 and GZ15 effectively degrade AFB1. Two new metabolites were identified and the key toxic lactone and furofuran rings both were destroyed and hydrogenated, meaning that lactonase and reductase might be involved in the degradation process.

Novel Aflatoxin-Degrading Enzyme from Bacillus shackletonii L7.

Food and feed contamination by aflatoxin (AF)B1 has adverse economic and health consequences. AFB1 degradation by microorganisms or microbial enzymes provides a promising preventive measure. To this end, the present study tested 43 bacterial isolates collected from maize, rice, and soil samples for AFB1-reducing activity. The higher activity was detected in isolate L7, which was identified as Bacillus shackletonii. L7 reduced AFB1, AFB2, and AFM1 levels by 92.1%, 84.1%, and 90.4%, respectively, after 72 h at 37 °C. The L7 culture supernatant degraded more AFB1 than viable cells and cell extracts; and the degradation activity was reduced from 77.9% to 15.3% in the presence of proteinase K and sodium dodecyl sulphate. A thermostable enzyme purified from the boiled supernatant was designated as Bacillus aflatoxin-degrading enzyme (BADE). An overall 9.55-fold purification of BADE with a recovery of 39.92% and an activity of 3.85 × 10³ U·mg-1 was obtained using chromatography on DEAE-Sepharose. BADE had an estimated molecular mass of 22 kDa and exhibited the highest activity at 70 °C and pH 8.0, which was enhanced by Cu2+ and inhibited by Zn2+, Mn2+, Mg2+, and Li⁺. BADE is the major protein involved in AFB1 detoxification. This is the first report of a BADE isolated from B. shackletonii, which has potential applications in the detoxification of aflatoxins during food and feed processing.

Toxin YafQ Reduces Escherichia coli Growth at Low Temperatures.

Toxin/antitoxin (TA) systems reduce metabolism under stress; for example, toxin YafQ of the YafQ/DinJ Escherichia coli TA system reduces growth by cleaving transcripts with in-frame 5'-AAA-G/A-3' sites, and antitoxin DinJ is a global regulator that represses its locus as well as controls levels of the stationary sigma factor RpoS. Here we investigated the influence on cell growth at various temperatures and found that deletion of the antitoxin gene, dinJ, resulted in both reduced metabolism and slower growth at 18°C but not at 37°C. The reduction in growth could be complemented by producing DinJ from a plasmid. Using a transposon screen to reverse the effect of the absence of DinJ, two mutations were found that inactivated the toxin YafQ; hence, the toxin caused the slower growth only at low temperatures rather than DinJ acting as a global regulator. Corroborating this result, a clean deletion of yafQ in the ΔdinJ ΔKmR strain restored both metabolism and growth at 18°C. In addition, production of YafQ was more toxic at 18°C compared to 37°C. Furthermore, by overproducing all the E. coli proteins, the global transcription repressor Mlc was found that counteracts YafQ toxicity only at 18°C. Therefore, YafQ is more effective at reducing metabolism at low temperatures, and Mlc is its putative target.

Variation in fungal microbiome (mycobiome) and aflatoxins during simulated storage of in-shell peanuts and peanut kernels.

Internal transcribed spacer 2 (ITS2) sequencing was used to characterize the peanut mycobiome during 90 days storage at five conditions. The fungal diversity in in-shell peanuts was higher with 110 operational taxonomic units (OTUs) and 41 genera than peanut kernels (91 OTUs and 37 genera). This means that the micro-environment in shell is more suitable for maintaining fungal diversity. At 20-30 d, Rhizopus, Eurotium and Wallemia were predominant in in-shell peanuts. In peanut kernels, Rhizopus (>30%) and Eurotium (>20%) were predominant at 10-20 d and 30 d, respectively. The relative abundances of Rhizopus, Eurotium and Wallemia were higher than Aspergillus, because they were xerophilic and grew well on substrates with low water activity (aw). During growth, they released metabolic water, thereby favoring the growth of Aspergillus. Therefore, from 30 to 90 d, the relative abundance of Aspergillus increased while that of Rhizopus, Eurotium and Wallemia decreased. Principal Coordinate Analysis (PCoA) revealed that peanuts stored for 60-90 days and for 10-30 days clustered differently from each other. Due to low aw values (0.34-0.72) and low levels of A. flavus, nine of 51 samples were contaminated with aflatoxins.

Ochratoxin A Producing Fungi, Biosynthetic Pathway and Regulatory Mechanisms.

Ochratoxin A (OTA), mainly produced by Aspergillus and Penicillum species, is one of the most important mycotoxin contaminants in agricultural products. It is detrimental to human health because of its nephrotoxicity, hepatotoxicity, carcinogenicity, teratogenicity, and immunosuppression. OTA structurally consists of adihydrocoumarin moiety linked with l-phenylalanine via an amide bond. OTA biosynthesis has been putatively hypothesized, although several contradictions exist on some processes of the biosynthetic pathway. We discuss recent information on molecular studies of OTA biosynthesis despite insufficient genetic background in detail. Accordingly, genetic regulation has also been explored with regard to the interaction between the regulators and the environmental factors. In this review, we focus on three aspects of OTA: OTA-producing strains, OTA biosynthetic pathway and the regulation mechanisms of OTA production. This can pave the way to assist in protecting food and feed from OTA contamination by understanding OTA biosynthetic pathway and regulatory mechanisms.