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.

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.

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.

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.

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.

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.

Switching micellization of pluronics in water by CO2.

The micellization of amphiphilic molecules is an interesting topic from both theoretical and practical points of view. Herein we have studied the effects of compressed CO(2) on the micellization of Pluronics in water by means of fluorescence, UV/Vis spectra, and small-angle X-ray scattering. It was found that CO(2) can induce the micellization of Pluronics in water, and the micelle can return to the initial state of molecular dispersion after depressurization. Therefore, the micellization of Pluronics in water can be switched through the easy control of pressure. Different from the common micelles with hydrophobic cores, interestingly, this CO(2)-induced micelle has an amphiphilic core, in which hydrophobic and hydrophilic domains coexist. On account of the ability to dissolve both polar and nonpolar components in the micellar core, the CO(2)-induced micelles can improve the reagent compatibilities frequently encountered in various applications. In an attempt to address this advantage, this micelle was utilized as template to the one-step synthesis of Au/silica core-shell composite nanoparticles. Furthermore, the underlying mechanism for the CO(2)-induced micellization of Pluronics in water was investigated by a series of experiments.

Water-in-oil-in-water double nanoemulsion induced by CO(2).

The cetyltrimethylammonium bromide (CTAB)/water/heptane emulsion system with different CO(2) pressure has been studied. The phase behavior investigation shows the nanoemulsion can be formed at suitable pressure range. The generalized indirect Fourier transformation (GIFT) analysis of the small-angle X-ray scattering (SAXS) data has drawn a clear picture of the structural information of the nanoemulsion, which reveals that the droplet of emulsion has a double structure with both the outer and inner droplet size in nanometre range. Furthermore, the investigation of the heptane/CTAB/water/CO(2) emulsion system by using electrical conductivity confirms the emulsion type transforms from O/W to W/O/W. In addition, the effect of different CTAB concentration on the nanoemulsion formation has been studied. It is found that enough CTAB concentration is necessary for the inclusion of continuous water into oil droplets. We also explored the application of the W/O/W double nanoemulsion in material synthesis. Interestingly, the hollow silica spheres with double shells were obtained in this CO(2)-induced double nanoemulsion.

Emulsion inversion induced by CO2.

Herein it was found that CO(2) could trigger an O/W to W/O emulsion inversion via a W/O/W nanoemulsion. In comparison with the conventionally used liquid or solid additives, the unusual advantage of this method is that the emulsion morphologies can be switched reversibly by the control of CO(2) pressure. Besides, CO(2) can be easily removed by depressurization and thus no extra separation process is needed, and CO(2) can be recycled. Furthermore, other lipophilic gases of ethylene, propylene and isobutane can also induce the O/W to W/O emulsion inversion. A possible mechanism for the gas-triggered emulsion inversion was proposed.

Carbon dioxide in ionic liquid microemulsions.

Tailor-made emulsion: a CO(2) -in-ionic-liquid microemulsion was produced for the first time. The CO(2)-swollen micelles are "tunable" because the micellar size can be easily adjusted by changing the pressure of CO(2). The microemulsion has potential applications in materials synthesis, chemical reactions, and extraction.

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.

Reversible switching of a micelle-to-vesicle transition by compressed CO2.

The study of the micelle-to-vesicle transition (MVT) is of great importance from both theoretical and practical points of view. Herein, we studied the effect of compressed CO(2) on the aggregation behavior of dodecyltrimethylammonium bromide (DTAB)/sodium dodecyl sulfate (SDS) mixed surfactants in aqueous solution by means of direct observation, turbidity and conductivity measurements, steady-state fluorescence, time-resolved fluorescence quenching (TRFQ), fluorescence quantum yield, and template methods. Interestingly, all these approaches showed that compressed CO(2) could induce the MVT in the surfactant system, and the vesicles returned to the micelles simply by depressurization; that is, CO(2) can be used to switch the MVT reversibly by controlling pressure. Some other gases, such as methane, ethylene, and ethane, could also induce the MVT of the surfactant solution. A possible mechanism is proposed on the basis of the packing-parameter theory and thermodynamic principles. It is shown that the mechanism of the MVT induced by a nonpolar gas is different from the MVT induced by polar and electrolyte additives.

Cylindrical-to-spherical shape transformation of lecithin reverse micelles induced by CO2.

The effect of CO(2) on the microstructure of L-alpha-phosphatidylcholine (lecithin) reverse micelles was studied. The small-angle X-ray scattering (SAXS) results show that CO(2) could induce a cylindrical-to-spherical micellar shape transformation. Fourier transform infrared (FT-IR) and UV-vis techniques were also utilized to investigate intermolecular interactions and micropolarity in the reverse micelles at different CO(2) pressures. The reduction of the degree of hydrogen bonding between surfactant headgroups and water with added CO(2) was found to be the main reason for the micellar shape transformation. In the absence of CO(2), the hydrogen bonding between water and P=O of lecithin forms a linking bridge in the interfacial layer. Therefore, the free movement of the polar head of lecithin is limited and the cylindrical reverse micelles are formed. Upon adding CO(2) to the reverse micelles, the hydrogen bonds between lecithin and water in reverse micelles are destroyed, which is favorable to forming spherical micelles. Moreover, the CO(2)-combined reverse micelles were utilized in the synthesis of silica particles. Rodlike silica nanoparticles were obtained in the absence of CO(2), and ellipsoidal and spherical mesoporous silica particles were formed in the presence of CO(2). This method of tuning micellar shape has many advantages compared to traditional methods.

Characterization of a thermostable xylanase from an alkaliphilic Bacillus sp.

A xylanase gene (xyn10) from alkaliphilic Bacillus sp. N16-5 was cloned and expressed in Pichia pastoris. The deduced amino acid sequence has 85% identity with xylanase xyn10A from B. halodurans and contains two potential N-glycosylation sites. The glycosylated Xyn10 with MW 48 kDa can hydrolyze birchwood and oatspelt xylan. The enzyme had optimum activity at pH 7 and 70°C, with the specific activity of 92.5U/mg. The Xyn10 retained over 90% residual activity at 60°C for 30 min but lost all activity at 80°C over 15 min. Most tested ions showed no or slight inhibition effects on enzyme activity.

[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.

Synthesis of uniform hollow silica spheres with ordered mesoporous shells in a CO2 induced nanoemulsion.

Monodisperse hollow silica spheres of uniform size and ordered mesoporous shells have been successfully fabricated in water/heptane/CTAB nanoemulsions induced by compressed CO(2).

[Recent advances and prospect on structural biology of beta-mannanase--a review].

Beta-mannanases (beta-1,4-D-mannanase, EC 3.2.1.78), as a hemicellulose hydrolase, are widely distributed in bacteria, fungi, plants and even animals. They can randomly hydrolyze the beta-1,4-mannosidic linkages in mannan and heteromannan and have great potential in the food/feed, pulp/paper, medicine, oil exploitation and detergent industries. Most beta-mannanases often display a modular organization and usually contain structurally discrete catalytic and non-catalytic modules. Catalytic domains of these enzymes share a (beta/alpha)8-barrel fold, which play important roles in substrate binding and catalysis. Carbohydrate binding modules, as the most common non-catalytic modules, fold as beta-sandwich and facilitate the targeting of these enzymes to polysaccharide. In this review, a brief introduction is given concerning structural characteristics and function of these beta-mannanase modules.

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 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 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.