Potential of Trichoderma spp. for Biocontrol of Aflatoxin-Producing Aspergillus flavus.

The inhibitory action of 20 antagonistic Trichoderma isolates against the aflatoxigenic isolate A. flavus ITEM 9 (Af-9) and their efficacy in reducing aflatoxin formation in vitro were examined. Production of metabolites with inhibitory effect by the Trichoderma isolates was also investigated. Antagonistic effect against Af-9 was assessed by inhibition of radial growth of the colonies and by fungal interactions in dual confrontation tests. A total of 8 out of 20 isolates resulted in a significant growth inhibition of 3-day-old cultures of Af-9, ranging from 13% to 65%. A total of 14 isolates reduced significantly the aflatoxin B1 (AfB1) content of 15-day-old Af-9 cultures; 4 were ineffective, and 2 increased AfB1. Reduction of AfB1 content was up to 84.9% and 71.1% in 7- and 15-day-old cultures, respectively. Since the inhibition of Af-9 growth by metabolites of Trichoderma was not necessarily associated with inhibition of AfB1 production and vice versa, we investigated the mechanism of reduction of AfB1 content at the molecular level by examining two strains: one (T60) that reduced both growth and mycotoxin content; and the other (T44) that reduced mycotoxin content but not Af-9 growth. The expression analyses for the two regulatory genes aflR and aflS, and the structural genes aflA, aflD, aflO and aflQ of the aflatoxin biosynthesis cluster indicated that neither strain was able to downregulate the aflatoxin synthesis, leading to the conclusion that the AfB1 content reduction by these Trichoderma strains was based on other mechanisms, such as enzyme degradation or complexation. Although further studies are envisaged to identify the metabolites involved in the biocontrol of A. flavus and prevention of aflatoxin accumulation, as well as for assessment of the efficacy under controlled and field conditions, Trichoderma spp. qualify as promising agents and possible alternative options to other biocontrol agents already in use.

New Insights of Transcriptional Regulator AflR in Aspergillus flavus Physiology.

Aspergillus flavus aflR, a gene encoding a Zn(II)2Cys6 DNA-binding domain, is an important transcriptional regulator of the aflatoxin biosynthesis gene cluster. Our previous results of Gene ontology (GO) analysis for the binding sites of AflR in A. flavus suggest that AflR may play an integrative regulatory role. In this study the ΔaflR and overexpression (OE) strains based on the well-established double-crossover recombinational technique were constructed to investigate the integrative function of the aflR gene in A. flavus. The disruption of aflR severely affected the aflatoxin biosynthetic pathway, resulting in a significant decrease in aflatoxin production. The aflatoxin B1 (AFB1) of the ΔaflR strain was 180 ng/mL and aflatoxin B2 (AFB2) was 2.95 ng/mL on YES medium for 5 days, which was 1/1,000 of that produced by the wild-type strain (WT). In addition, the ΔaflR strain produced relatively sparse conidia and a very small number of sclerotia. On the seventh day, the sclerotia yield on each plate of the WT and OE strains exceeded 1,000, while the sclerotial formation of the ΔaflR strain was not detected until 14 days. However, the biosynthesis of cyclopiazonic acid (CPA) was not affected by aflR gene disruption. Transcriptomic analysis of the ΔaflR strain grown on potato dextrose agar (PDA) plates at 0 h, 24 h, and 72 h showed that expression of clustering genes involved in the biosynthesis of aflatoxin was significantly downregulated. Meanwhile, the ΔaflR strain compared with the WT strain showed significant expression differences in genes involved in spore germination, sclerotial development, and carbohydrate metabolism compared to the WT. The results demonstrated that the A. flavus aflR gene also played a positive role in the fungal growth and development in addition to aflatoxin biosynthesis. IMPORTANCE Past studies of the A. flavus aflR gene and its orthologues in related Aspergillus species were solely focused on their roles in secondary metabolism. In this study, we used the ΔaflR and OE strains to demonstrate the role of aflR in growth and development of A. flavus. For the first time, we confirmed that the ΔaflR strain also was defective in production of conidia and sclerotia, asexual propagules of A. flavus. Our transcriptomic analysis further showed that genes involved in spore germination, sclerotial development, aflatoxin biosynssssthesis, and carbohydrate metabolism exhibited significant differences in the ΔaflR strain compared with the WT strain. Our study indicates that AflR not only plays an important role in regulating aflatoxin synthesis but also in playing a positive role in the conidial formation and sclerotial development in A. flavus. This study reveals the critical and positive role of the aflR gene in fungal growth and development, and provides a theoretical basis for the genetic studies of other aspergilli.

Preservation of Mimosa tenuiflora Antiaflatoxigenic Activity Using Microencapsulation by Spray-Drying.

Mimosa tenuiflora aqueous extract (MAE) is rich in phenolic compounds. Among them, condensed tannins have been demonstrated to exhibit a strong antioxidant and antiaflatoxin B1 activities in Aspergillus flavus. Since antioxidant capacity can change with time due to environmental interactions, this study aimed to evaluate the ability of encapsulation by spray-drying of Mimosa tenuiflora aqueous extract to preserve their biological activities through storage. A dry formulation may also facilitate transportation and uses. For that, three different wall materials were used and compared for their efficiency. Total phenolic content, antioxidant activity, antifungal and antiaflatoxin activities were measured after the production of the microparticles and after one year of storage at room temperature. These results confirmed that encapsulation by spray-drying using polysaccharide wall materials is able to preserve antiaflatoxin activity of Mimosa tenuiflora extract better than freezing.

Fusarium verticillioides and Aspergillus flavus Co-Occurrence Influences Plant and Fungal Transcriptional Profiles in Maize Kernels and In Vitro.

Climate change will increase the co-occurrence of Fusarium verticillioides and Aspergillus flavus, along with their mycotoxins, in European maize. In this study, the expression profiles of two pathogenesis-related (PR) genes and four mycotoxin biosynthetic genes, FUM1 and FUM13, fumonisin pathway, and aflR and aflD, aflatoxin pathway, as well as mycotoxin production, were examined in kernels and in artificial medium after a single inoculation with F. verticillioides or A. flavus or with the two fungi in combination. Different temperature regimes (20, 25 and 30 °C) over a time-course of 21 days were also considered. In maize kernels, PR genes showed the strongest induction at 25 °C in the earlier days post inoculation (dpi)with both fungi inoculated singularly. A similar behaviour was maintained with fungi co-occurrence, but with enhanced defence response at 9 dpi under 20 °C. Regarding FUM genes, in the kernels inoculated with F. verticillioides the maximal transcript levels occurred at 6 dpi at 25 °C. At this temperature regime, expression values decreased with the co-occurrence of A. flavus, where the highest gene induction was detected at 20 °C. Similar results were observed in fungi grown in vitro, whilst A. flavus presence determined lower levels of expression along the entire time-course. As concerns afl genes, considering both A. flavus alone and in combination, the most elevated transcript accumulation occurred at 30 °C during all time-course both in infected kernels and in fungi grown in vitro. Regarding mycotoxin production, no significant differences were found among temperatures for kernel contamination, whereas in vitro the highest production was registered at 25 °C for aflatoxin B1 and at 20 °C for fumonisins in the case of single inoculation. In fungal co-occurrence, both mycotoxins resulted reduced at all the temperatures considered compared to the amount produced with single inoculation.

Mimosa tenuiflora Aqueous Extract: Role of Condensed Tannins in Anti-Aflatoxin B1 Activity in Aspergillus flavus.

Aflatoxin B1 (AFB1) is a potent carcinogenic mycotoxin that contaminates numerous crops pre- and post-harvest. To protect foods and feeds from such toxins without resorting to pesticides, the use of plant extracts has been increasingly studied. The most interesting candidate plants are those with strong antioxidative activity because oxidation reactions may interfere with AFB1 production. The present study investigates how an aqueous extract of Mimosa tenuiflora bark affects both the growth of Aspergillus flavus and AFB1 production. The results reveal a dose-dependent inhibition of toxin synthesis with no impact on fungal growth. AFB1 inhibition is related to a down-modulation of the cluster genes of the biosynthetic pathway and especially to the two internal regulators aflR and aflS. Its strong anti-oxidative activity also allows the aqueous extract to modulate the expression of genes involved in fungal oxidative-stress response, such as msnA, mtfA, atfA, or sod1. Finally, a bio-guided fractionation of the aqueous extract demonstrates that condensed tannins play a major role in the anti-aflatoxin activity of Mimosa tenuiflora bark.

Inhibition of Aspergillus flavus growth and aflatoxins production on peanuts over α-Fe2O3 nanorods under sunlight irradiation.

Peanut is an important resource of edible oil and digestible protein in daily life, which is rich in the nutriments and antioxidants such as vitamins, minerals and polyphenols. However, peanut is susceptible to the contamination of Aspergillus flavus (A. flavus), which can produce highly carcinogenic toxins that brings serious threats to human health and food safety. Exploring green and effective methods to control A. flavus is meaningful. Herein, a green and economical way to control A. flavus on peanuts was demonstrated. It was found that the growth of A. flavus hyphae and germination of its spores could be inhibited in the presence of α-Fe2O3 nanorods under sunlight irradiation according to the agar diffusion method, flat colony counting method and fluorescence-based live/dead test. The diameter of inhibition zone was 22.3 ± 0.2 mm and the inhibition rate of spores germination was about 60 ± 5%, when the concentration of α-Fe2O3 was 10 mg/mL for 7 h sunlight irradiation. Most important, α-Fe2O3 showed the photocatalytic inhibition of A. flavus on peanuts under sunlight irradiation with the inhibition rate of about 90 ± 5%, and the production of aflatoxin B1 and aflatoxin B2 were reduced by 90 ± 2% and 70 ± 3%, respectively. By comparing the fat contents, protein contents, acid value, peroxide value and antioxidative compositions of peanuts, it was found that there was no obvious effect on the quality of peanuts after inhibition treatment. The findings provide a green, safe and economical strategy to control A. flavus on peanuts, which may be as a promising way to be used in food and agro-food preservation.

Aspergillus flavus Growth Inhibition and Aflatoxin B1 Decontamination by Streptomyces Isolates and Their Metabolites.

Aflatoxin B1 is a potent carcinogen produced by Aspergillus flavus, mainly during grain storage. As pre-harvest methods are insufficient to avoid mycotoxin presence during storage, diverse curative techniques are being investigated for the inhibition of fungal growth and aflatoxin detoxification. Streptomyces spp. represent an alternative as they are a promising source of detoxifying enzymes. Fifty-nine Streptomyces isolates and a Streptomyces griseoviridis strain from the commercial product Mycostop®, evaluated against Penicillium verrucosum and ochratoxin A during previous work, were screened for their ability to inhibit Aspergillus flavus growth and decrease the aflatoxin amount. The activities of bacterial cells and cell-free extracts (CFEs) from liquid cultures were also evaluated. Fifty-eight isolates were able to inhibit fungal growth during dual culture assays, with a maximal reduction going down to 13% of the control. Aflatoxin-specific production was decreased by all isolates to at least 54% of the control. CFEs were less effective in decreasing fungal growth (down to 40% and 55% for unheated and heated CFEs, respectively) and aflatoxin-specific production, with a few CFEs causing an overproduction of mycotoxins. Nearly all Streptomyces isolates were able to degrade AFB1 when growing in solid and liquid media. A total degradation of AFB1 was achieved by Mycostop® on solid medium, as well as an almost complete degradation by IX20 in liquid medium (6% of the control). CFE maximal degradation went down to 37% of the control for isolate IX09. The search for degradation by-products indicated the presence of a few unknown molecules. The evaluation of residual toxicity of the tested isolates by the SOS chromotest indicated a detoxification of at least 68% of AFB1's genotoxicity.

Impacts of Climate Change Interacting Abiotic Factors on Growth, aflD and aflR Gene Expression and Aflatoxin B1 Production by Aspergillus flavus Strains In Vitro and on Pistachio Nuts.

Pistachio nuts are an important economic tree nut crop which is used directly or processed for many food-related activities. They can become colonized by mycotoxigenic spoilage fungi, especially Aspergillus flavus, mainly resulting in contamination with aflatoxins (AFs), especially aflatoxin B1 (AFB1). The prevailing climate in which these crops are grown changes as temperature and atmospheric CO2 levels increase, and episodes of extreme wet/dry cycles occur due to human industrial activity. The objectives of this study were to evaluate the effect of interacting Climate Change (CC)-related abiotic factors of temperature (35 vs. 37 °C), CO2 (400 vs. 1000 ppm), and water stress (0.98-0.93 water activity, aw) on (a) growth (b) aflD and aflR biosynthetic gene expression and (c) AFB1 production by two strains A. flavus (AB3, AB10) in vitro on milled pistachio-based media and when colonizing layers of shelled raw pistachio nuts. The A. flavus strains were resilient in terms of growth on pistachio-based media and the colonisation of pistachio nuts with no significant difference when exposed to the interacting three-way climate-related abiotic factors. However, in vitro studies showed that AFB1 production was significantly stimulated (p < 0.05), especially when exposed to 1000 ppm CO2 at 0.98-0.95 aw and 35 °C, and sometimes in the 37 °C treatment group at 0.98 aw. The relative expression of the structural aflD gene involved in AFB1 biosynthesis was decreased or only slightly increased, relative to the control conditions at elevated CO, regardless of the aw level examined. For the regulatory aflR gene expression, there was a significant (p < 0.05) increase in 1000 ppm CO2 and 37 °C for both strains, especially at 0.95 aw. The in situ colonization of pistachio nuts resulted in a significant (p < 0.05) stimulation of AFB1 production at 35 °C and 1000 ppm CO2 for both strains, especially at 0.98 aw. At 37 °C, AFB1 production was either decreased, in strain AB3, or remained similar, as in strain AB10, when exposed to 1000 ppm CO2. This suggests that CC factors may have a differential effect, depending on the interacting conditions of temperature, exposure to CO2 and the level of water stress on AFB1 production.

Cross-talk between Fusarium verticillioides and Aspergillus flavus in vitro and in planta.

Driven by increasing temperatures and the higher incidences of heat waves during summer, an increased incidence of Aspergillus flavus next to Fusarium verticillioides in European maize can be expected. In the current study, we investigated the interaction between both species. Colonies of A. flavus/F. verticillioides were grown in a single culture, in a dual culture, and in a mixed culture. The growth rate of A. flavus and F. verticillioides grown in a dual or mixed culture with the other species was clearly slower compared to the growth rate in a single culture. Mycotoxin production was in most cases negatively affected by dual or mixed inoculation. In planta, a dual inoculation resulted in reduced lesions of A. flavus, whereas the lesion size and toxin production of F. verticillioides were unaffected in the presence of A. flavus. The lesions as a result of a mixed inoculation were 112% bigger than a single A. flavus inoculation and 9% smaller than a single F. verticillioides inoculation. The fumonisin levels were 17% higher compared to a single inoculation. In case A. flavus was present two days before F. verticillioides, the lesion size of F. verticillioides was 55% smaller compared to a single F. verticillioides inoculation, and fumonisin production was almost completely inhibited. The interaction between A. flavus and F. verticillioides is highly dynamic and depends on the experimental conditions, on the variables measured and on the way they colonize the host, in two inoculation points, simultaneously in one inoculation point, or sequentially one species colonizing an existing lesion made by the other.

Changes in Bacterial Endophyte Community Following Aspergillus flavus Infection in Resistant and Susceptible Maize Kernels.

Aspergillus flavus (A. flavus)-mediated aflatoxin contamination in maize is a major global economic and health concern. As A. flavus is an opportunistic seed pathogen, the identification of factors contributing to kernel resistance will be of great importance in the development of novel mitigation strategies. Using V3-V4 bacterial rRNA sequencing and seeds of A. flavus-resistant maize breeding lines TZAR102 and MI82 and a susceptible line, SC212, we investigated kernel-specific changes in bacterial endophytes during infection. A total of 81 bacterial genera belonging to 10 phyla were detected. Bacteria belonging to the phylum Tenericutes comprised 86-99% of the detected phyla, followed by Proteobacteria (14%) and others (<5%) that changed with treatments and/or genotypes. Higher basal levels (without infection) of Streptomyces and Microbacterium in TZAR102 and increases in the abundance of Stenotrophomonas and Sphingomonas in MI82 following infection may suggest their role in resistance. Functional profiling of bacteria using 16S rRNA sequencing data revealed the presence of bacteria associated with the production of putative type II polyketides and sesquiterpenoids in the resistant vs. susceptible lines. Future characterization of endophytes predicted to possess antifungal/ anti-aflatoxigenic properties will aid in their development as effective biocontrol agents or microbiome markers for maize aflatoxin resistance.

Predicted Aflatoxin B1 Increase in Europe Due to Climate Change: Actions and Reactions at Global Level.

Climate change (CC) is predicted to increase the risk of aflatoxin (AF) contamination in maize, as highlighted by a project supported by EFSA in 2009. We performed a comprehensive literature search using the Scopus search engine to extract peer-reviewed studies citing this study. A total of 224 papers were identified after step I filtering (187 + 37), while step II filtering identified 25 of these papers for quantitative analysis. The unselected papers (199) were categorized as "actions" because they provided a sounding board for the expected impact of CC on AFB1 contamination, without adding new data on the topic. The remaining papers were considered as "reactions" of the scientific community because they went a step further in their data and ideas. Interesting statements taken from the "reactions" could be summarized with the following keywords: Chain and multi-actor approach, intersectoral and multidisciplinary, resilience, human and animal health, and global vision. In addition, fields meriting increased research efforts were summarized as the improvement of predictive modeling; extension to different crops and geographic areas; and the impact of CC on fungi and mycotoxin co-occurrence, both in crops and their value chains, up to consumers.

Impact of encapsulated orange essential oil with ß-cyclodextrin on technological, digestibility, sensory properties of wheat cakes as well as Aspergillus flavus spoilage.

BACKGROUND: The majority of studies with essential oils in foods focus mainly on improving the shelf life of products; however, the present study goes further and demonstrates not only the effect of essential oil on conservation properties, but also the effect of free and encapsulated orange essential oil (OEO) on the technological, sensorial and digestibility properties of bakery products. RESULTS: OEO was encapsulated into ß-cyclodextrin (ß-CD) by inclusion complex formation (ß-CD/OEO 97.4% of encapsulation efficiency). OEO demonstrated in vitro antifungal activity against Aspergillus flavus (inhibition zone of 11.33 mm on mycelial growth). In situ antifungal activity against A. flavus confirmed that free OEO can effectively delay the fungal growth, unlike encapsulated OEO. Regarding texture profile and starch digestibility: cake with ß-CD/OEO showed lower hardness (31.64 N) and lower starch digestibility (69.10%) than cake with free OEO (44.30 N; 82.10%, respectively) and the addition of OEO (both free and encapsulated) decreased the adhesiveness of the cakes. Cake with free OEO showed a higher intensity of orange aroma, being preferred by 60% of panelists, whereas cake with ß-CD/OEO presented a very slight orange taste and aroma. CONCLUSION: The encapsulation of OEO into ß-CD improved the crumb texture of cakes and promoted a lower starch digestibility in the cakes. On the other hand, the encapsulation process was not effective under the conditions tested (OEO concentration and baking temperatures), compromising the action of the OEO as a natural flavoring and preservative agent. © 2021 Society of Chemical Industry.

Effect of Temperature during Drying and Storage of Dried Figs on Growth, Gene Expression and Aflatoxin Production.

Dried fig is susceptible to infection by Aspergillus flavus, the major producer of the carcinogenic mycotoxins. This fruit may be contaminated by the fungus throughout the entire chain production, especially during natural sun-drying, post-harvest, industrial processing, storage, and fruit retailing. Correct management of such critical stages is necessary to prevent mould growth and mycotoxin accumulation, with temperature being one of the main factors associated with these problems. The effect of different temperatures (5, 16, 25, 30, and 37 °C) related to dried-fig processing on growth, one of the regulatory genes of aflatoxin pathway (aflR) and mycotoxin production by A. flavus, was assessed. Firstly, growth and aflatoxin production of 11 A. flavus strains were checked before selecting two strains (M30 and M144) for in-depth studies. Findings showed that there were enormous differences in aflatoxin amounts and related-gene expression between the two selected strains. Based on the results, mild temperatures, and changes in temperature during drying and storage of dried figs should be avoided. Drying should be conducted at temperatures >30 °C and close to 37 °C, while industry processing, storage, and retailing of dried figs are advisable to perform at refrigeration temperatures (<10 °C) to avoid mycotoxin production.

Synthetic antimicrobial agents inhibit aflatoxin production.

Antimicrobial peptides (AMPs) are biologically active molecules that can eradicate bacteria by destroying the bacterial membrane structure, causing the bacteria to rupture. However, little is known about the extent and effect of AMPs on filamentous fungi. In this study, we synthesized small molecular polypeptides by an inexpensive heat conjugation approach and examined their effects on the growth of Aspergillus flavus and its secondary metabolism. The antimicrobial agents significantly inhibited aflatoxin production, conidiation, and sclerotia formation in A. flavus. Furthermore, we found that the expression of aflatoxin structural genes was significantly inhibited, and the intracellular reactive oxygen species (ROS) level was reduced. Additionally, the antimicrobial agents can change membrane permeability. Overall, our results demonstrated that antimicrobial agents, safe to mammalian cells, have an obvious impact on aflatoxin production, which indicated that antimicrobial agents may be adopted as a new generation of potential agents for controlling aflatoxin contamination.

Eugenol loaded chitosan nanoemulsion for food protection and inhibition of Aflatoxin B1 synthesizing genes based on molecular docking.

The present investigation entails the fabrication and characterization of nanometric emulsion of eugenol (Nm-eugenol) encompassed into chitosan for assessing bio-efficacy in terms of in vitro antifungal actions, antiaflatoxigenic potential, and in situ preservative efficacy against Aspergillus flavus infestation and aflatoxin B1 (AFB1) mediated loss of dietary minerals, lipid triglycerides and alterations in composition of important macronutrients in stored rice. Nm-eugenol characterized by SEM, XRD, and FTIR exhibited biphasic burst release of eugenol. Reduction in ergosterol and methylglyoxal (AFB1-inducer) content after Nm-eugenol fumigation depicted biochemical mechanism of antifungal and antiaflatoxigenic activities. In silico 3D homology docking of eugenol with Ver-1 gene validated molecular mechanism of AFB1 inhibition. Further, significant protection of rice seeds from fungi, AFB1 contamination and preservation against loss of rice minerals, macronutrients and lipids during storage suggested deployment of chitosan as a biocompatible wall material for eugenol encapsulation and application as novel green preservative for food protection.

Computational modelling of survival of Aspergillus flavus in peanut kernels during hot air-assisted radio frequency pasteurization.

In recent years, radio frequency (RF) heating is getting popular as an alternative pasteurization method for agricultural commodities and low moisture foods. Computer simulation is an effective way to help understand RF interactions with food components and predict temperature distributions among food samples after RF treatments. In this study, a computer model based on Joule heating and thermal inactivation kinetic of A. flavus was established to predict both temperature distribution and microbial reduction among peanut kernels after RF processing. For the process validation, three 2-g peanut samples inoculated with 40 µL A. flavus were placed at three representative locations among 2.17 kg peanut kernels and subjected to various processing conditions in a 27.12 MHz, 6 kW RF heating unit together with hot air system. Results showed that the average difference of the sample temperature and microbial reduction between simulation and experiment was small with RMSE values of 0.009 °C and 0.012 °C, and 0.31 log CFU/g and 0.42 log CFU/g for peanut moisture contents of 7.56% and 12.02% w. b., respectively. Nonuniform RF heating resulted in the least lethality of A. flavus at the cold spot. The validated computer model was further used to estimate microbial reduction distributions at other target temperatures based on predicted temperature profiles. This computer model may help design the RF pasteurization protocols for peanut kernels without extensive experiments in food industry.

High voltage atmospheric cold plasma treatment inactivates Aspergillus flavus spores and deoxynivalenol toxin.

Fungal contamination is a concern for the food industry. Fungal spores resist food sterilization treatments and produce mycotoxins that are toxic for animals and humans. Technologies that deactivate spores and toxins without impacting food quality are desirable. This study demonstrates the efficiency of a high voltage atmospheric cold plasma (HVACP) technology using air to generate reactive oxygen (ROS) and nitrogen (RNS) species for the degradation of Aspergillus flavus cultures and the deoxynivalenol (DON) mycotoxin. Optical emission and absorption spectroscopy demonstrate ionization of hydroxyl groups, atomic oxygen and nitrogen, and confirm production of ROS and RNS, e.g. O3, NO2, NO3, N2O4, and N2O5. Fungal cultures show a depletion in pigmentation and an ~50% spore inactivation after 1-min treatments. Treated spores show surface ablation and membrane degradation by scanning electron microscopy. Twenty-minute direct HVACP treatments of 100 µg of DON in one mL aqueous suspensions resulted in a greater than 99% reduction in DON structure and rescued over 80% of Caco-2 cell viability; however, the same treatment on 100 µg of powdered DON toxin only showed a 33% reduction in DON and only rescued 15% of cell viability. In summary, HVACP air treatment can inactivate both fungal spores and toxins in minutes.

Effect of different carbon sources on the growth and enzyme production of a toxigenic and a non-toxigenic strain of Aspergillus flavus.

Two strains of A. flavus one toxigenic (CECT 2687) and the other non-toxigenic (NRRL 6541) were studied for their genomic potential, growth capacity, and the production of enzymes on simple sugars, polysaccharides, and complex substrates under solid-state fermentation (SSF). According to the genome analysis, this fungus has many genes to degrade different types of polysaccharides and therefore it would be able to grow on different substrates. Both strains grow in all the carbon sources, but visibly CECT2687 grows slower than NRRL6541. However, we propose the growth index (GI) to establish a dry weight-diameter relationship as a more reliable measure that truly shows the growth preferences of the fungus. Considering this, the NRRL6541 shows less growth in 11 of the 16 evaluated carbon sources than CECT2687. Complex substrates were the best carbon source for the growth of both strains. Corncob (CC) induced the production of xylanases, pectinases, and almost all the accessory enzymes evaluated (except for α-xylosidase) this could make it an agricultural waste of interest to produce hemicellulolytic enzymes. Both strains produce a great variety of xylanases and pectinases (pathogenicity factors) making A. flavus a good potential candidate for the degradation of polysaccharides with a high content of xylan and pectin.

Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus.

Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na+K+-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

Synthesis, characterization, antimicrobial, antioxidant and computational evaluation of N-acyl-morpholine-4-carbothioamides.

The present research paper reports the convenient synthesis, successful characterization, in vitro antibacterial, antifungal, antioxidant potency and biocompatibility of N-acyl-morpholine-4-carbothioamides (5a-5j). The biocompatible derivatives were found to be highly active against the tested bacterial and fungal strains. Moreover, some of the screened N-acyl-morpholine-4-carbothioamides exhibited excellent antioxidant potential. Docking simulation provided additional information about possibilities of their inhibitory potential against RNA. It has been predicted by in silico investigation of the binding pattern that compounds 5a and 5j can serve as the potential surrogate for design of novel and potent antibacterial agents. The results for the in vitro bioassays were promising with the identification of compounds 5a and 5j as the lead and selective candidate for RNA inhibition. Results of the docking computations further ascertained the inhibitory potential of compound 5a. Based on the in silico studies, it can be suggested that compounds 5a and 5j can serve as a structural model for the design of antibacterial agents with better inhibitory potential. Binding mode of compound 5j inside the active site of RNA in 3D space. 5j displayed highest antibacterial potential than the reference drug ampicillin with ZOI 10.50 mm against Staphylococcus aureus. 5j also displayed highest antifungal potential than the reference drug amphotericin B with ZOI 18.20 mm against Fusarium solani.