Food crops around the world are commonly contaminated with Aspergillus flavus, which can produce the carcinogenic mycotoxin aflatoxin B1 (AFB1). The objective of this study is to test an X-ray irradiation sterilization method for studying AFB1 in contaminated maize samples in the laboratory. Maize that had been naturally contaminated with 300 ppb AFB1 by the growth of aflatoxigenic A. flavus was ground and then irradiated at 0.0, 1.0, 1.5, 2.0, 2.5, and 3.0 kGy. A. flavus was quantified by dilution plating on potato dextrose agar (PDA) and modified Rose Bengal media (MDRB) for viability and qPCR for gene presence. AFB1 was quantified by HPLC and ELISA. A. flavus viability, but not gene copies, significantly decreased with increasing doses of radiation (PDA: p < 0.001; MDRB: p < 0.001; qPCR: p = 0.026). AFB1 concentration did not significantly change with increasing doses of radiation (HPLC: p = 0.153; ELISA: p = 0.567). Our results imply that X-ray irradiation is an effective means of reducing viable A. flavus without affecting AFB1 concentrations. Reducing the hazard of fungal spores and halting AFB1 production at the targeted dose are important steps to safely and reproducibly move forward research on the global mycotoxin challenge.

We evaluated the specificity of the primers OPF-01, P54, and 1253 to identify A. fumigatus, A. flavus, and A. niger, respectively, with the RAPD-PCR method. Eighty-two isolates belonging to the sections Fumigati, Flavi, and Nigri were used. The isolates were identified by phenotypic (macro- and micromorphology) and genotypic (partial sequences of the BenA gene) methods. The RAPD-PCR method was used to obtain polymorphic patterns with the primers OPF-01, P54, and 1253. The specificity of the polymorphic patterns of the isolates of each species was evaluated through the UPGMA clustering method and logistic regression model. All isolates of the genus Aspergillus were identified at the section level by macro- and micromorphology showing the typical morphology of the sections Fumigati, Flavi, and Nigri, and the species were identified by the construction of the phylogeny of the partial sequence of the BenA gene. The patterns\' polymorphic strains obtained with the primers OPF-01, P54, and 1253 for the isolates of A. fumigatus, A. flavus, and A niger, respectively, showed the same polymorphic pattern as the reference strains for each species. To verify the specificity of the primers, they were tested with other species from the sections Fumigati, Flavi and Nigri. The results support that the primers OPF-01, P54, and 1253 generate polymorphic patterns by RAPD-PCR species specific to A. fumigatus, A. flavus, and A. niger, respectively.

Essential oils possess significant antimicrobial and antioxidant properties and are increasingly used as natural substitutes for food preservation. Therefore, this study investigated the potential application of rosemary essential oil (REO) and REO nano-emulsion in the dairy plant. The antimicrobial effects of REO and REO nano-emulsion were determined by an agar well diffusion assay after chemical profiling by Gas Chromatography-Mass Spectrometry (GC-MS). The REO nano-emulsion was characterized by a Transmission Electron Microscope (TEM). The REO chemical profile revealed the presence of 42 chemical compounds, including 1, 8-cineole (9.72 %), and α-pinene (5.46 %) as major active components. REO nano-emulsion demonstrated significant antimicrobial activity compared to REO (P < 0.05) with a MIC value of 0.0001 mg/ml against Listeria monocytogenes and Aspergillus flavus and 0.001 mg/ml against Pseudomonas aeruginosa and Bacillus cereus. REO nano-emulsion enhanced the oxidative stability of pasteurized fresh cream, revealing a non-significant difference compared with that inoculated with butylated hydroxy anisol (BHA; synthetic antioxidant) (P˃ 0.05). Fortified cream and Karish cheese with REO nano-emulsion were evaluated organoleptically, and the results showed higher grades of overall acceptability when compared to control samples with a statistically significant difference (P < 0.05). Viability studies were estimated using the previously mentioned microorganisms in fortified fresh cream and Karish cheese with REO nano-emulsion. Results of the fortified cream showed a complete reduction of L. monocytogenes, A. flavus, and B. cereus on days 5, 7, and 10, respectively, and a 96.93 % reduction of P. aeruginosa by the end of the storage period. Regarding Karish cheese viability studies, C. albicans, A. flavus, and P. aeruginosa exhibited complete reduction on days 10, 10, and 15 of storage, respectively. In conclusion, REO nano-emulsion was recommended as a natural, safe, and effective antimicrobial and antioxidant additive in the dairy industry.

Phenylalanine ammonia-lyase (PAL) is an essential enzyme in the phenylpropanoid pathway, in which numerous aromatic intermediate metabolites play significant roles in plant growth, adaptation, and disease resistance. Cultivated peanuts are highly susceptible to Aspergillus flavus L. infection. Although PAL genes have been characterized in various major crops, no systematic studies have been conducted in cultivated peanuts, especially in response to A. flavus infection. In the present study, a systematic genome-wide analysis was conducted to identify PAL genes in the Arachis hypogaea L. genome. Ten AhPAL genes were distributed unevenly on nine A. hypogaea chromosomes. Based on phylogenetic analysis, the AhPAL proteins were classified into three groups. Structural and conserved motif analysis of PAL genes in A. hypogaea revealed that all peanut PAL genes contained one intron and ten motifs in the conserved domains. Furthermore, synteny analysis indicated that the ten AhPAL genes could be categorized into five pairs and that each AhPAL gene had a homologous gene in the wild-type peanut. Cis-element analysis revealed that the promoter region of the AhPAL gene family was rich in stress- and hormone-related elements. Expression analysis indicated that genes from Group I (AhPAL1 and AhPAL2), which had large number of ABRE, WUN, and ARE elements in the promoter, played a strong role in response to A. flavus stress.

Aflatoxins from the fungus Aspergillus flavus (A. flavus) that contaminate stored peanuts is a major hazard to human health worldwide. Reducing A. flavus in soil can decrease the risk of aflatoxins in stored peanuts. In this experiment, we determined whether peanuts grown on soil fumigated with dazomet (DZ), metham sodium (MS), allyl isothiocyanate (AITC), chloropicrin (PIC) or dimethyl disulfide (DMDS) would reduce of the quantity of A. flavus and its toxin\'s presence. The results of bioassays and field tests showed that PIC was the most effective fumigant for preventing and controlling A. flavus, followed by MS. PIC and MS applied to the soil for 14 d resulted in LD50 values against A. flavus of 3.558 and 4.893 mg kg-1, respectively, leading to almost 100% and 98.82% effectiveness of A. flavus, respectively. Peanuts harvested from fumigated soil and then stored for 60 d resulted in undetectable levels of aflatoxin B1 (AFB1) compared to unfumigated soil that contained 0.64 ug kg-1 of AFB1, which suggested that soil fumigation can reduce the probability of aflatoxin contamination during peanut storage and showed the potential to increase the safety of peanuts consumed by humans. Further research is planned to determine the practical value of our research in commercial practice.

This study identified and monitored the levels of aflatoxins (B1 and B2) produced by Aspergillus flavus isolate VKMN22 (OP355447) in maize samples sourced from a local shop in Johannesburg, South Africa. Maize samples underwent controlled incubation after initial rinsing, and isolates were identified through morphological and molecular methods. In another experiment, autoclaved maize grains were intentionally re-inoculated with the identified fungal isolate using spore suspension (106 spore/mL), after which 1 g of the contaminated maize sample was inoculated on PDA media and cultured for seven days. The aflatoxin concentrations in the A. flavus contaminated maize inoculated on culture media was monitored over seven weeks and then measured using liquid chromatography-mass spectroscopy (LC-MS). Results confirmed the successful isolation of A. flavus strain VKMN22 with accession number OP355447, which consistently produced higher levels of AFB1 compared to AFB2. AF concentrations increased from week one to five, then declined in week six and seven. AFB1 levels ranged from 594.3 to 9295.33 µg/kg (week 1-5) and then reduced from 5719.67 to 2005 µg/kg in week six and seven), while AFB2 levels ranged from 4.92 to 901.67 µg/kg (weeks 1-5) and then degraded to 184 µg/kg in week six then 55.33 µg/kg (weeks 6-7). Levene\'s tests confirmed significantly higher mean concentrations of AFB1 compared to AFB2 (p ≤ 0.005). The study emphasizes the importance of consistent biomonitoring for a dynamic understanding of AF contamination, informing accurate prevention and control strategies in agricultural commodities thereby safeguarding food safety.

Aureobasidium melanogenum was found to be grown the best at the constant pH 7.0 and to produce the highest amount of liamocins at the constant pH 3.0. Therefore, the wild type strain A. melanogenum 9-1 and the engineered strain V33 constructed in the laboratory were grown at the constant pH 7.0 for 48 h, then, they were continued to be cultivated at the constant pH 3.0. Under such conditions, A. melanogenum 9-1 produced 36.51 ± 0.55 g L-1 of liamocin and its cell mass was 27.43 ± 0.63 and 6.00 ± 0.11 g L-1 of glucose was left in the finished medium within 168 h while the engineered strain V33 secreted 70.86 ± 2.04 g L-1 of liamocin, its cell mass was 31.63 ± 0.74 g L-1 , 0.16 ± 0.01 g L-1 of glucose was maintained in the finished medium. Then, Massoia lactone was released from the produced liamocins. The released Massoia lactone loaded in the nanoemulsions could be used to actively damage cell wall and cell membrane of both spores and mycelia of Aspergillus flavus, leading to its cell necrosis. Massoia lactone loaded in the nanoemulsions also actively inhibited cell growth of A. flavus, its conidia production and aflatoxin biosynthesis on peanuts, indicating that Massoia lactone loaded in the nanoemulsions had highly potential application in controlling cell growth of A. flavus and aflatoxin biosynthesis in foods and feedstuffs.

Silver nanoparticles (AgNPs), which have recently gained attention due to their antimicrobial activity, can also be produced by green synthesis. The aims of this study were to (i) characterise green synthesized AgNPs using microwave-assisted aqueous extracts of Galium aparine (G-AgNPs) and Helichrysum arenarium (H-AgNPs) and (ii) investigate the combined antimicrobial effects of the G- and H-AgNPs in different ratios. Nanoparticle formation and reactions were determined with UV-Vis spectroscopy. The G-AgNPs were 52.0±10.9 nm in size, with a 0.285±0.034 polydispersity index (PDI), and a -17.9±0.9 mV zeta potential. For H-AgNPs these characteristics were 23.9±1.0 nm, 0.280±0.032, and -21.3±2.7 mV, respectively. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) confirmed that the particles were monodisperse and spherical. The Fourier transform-infrared spectroscopy (FT-IR) results showed the presence of reducing agents that stabilised the AgNPs. Three different nanoformulations (NF-1, NF-2, and NF-3) were prepared by combining these two synthesised nanoparticles in different ratios and their antimicrobial activity was tested against E. coli, S. aureus, C. albicans, and A. flavus. Our study is the first to show that combining AgNPs from two different biological sources can produce effective nanoformulations with improved antibacterial activity against E. coli and S. aureus. These nanoformulations showed lower minimum inhibitory concentrations (31.25 µg/mL against E. coli with all NFs; 62.5 µg/mL for NF-1 and 125 µg/mL for NF-2/3 against S. aureus) than G-AgNPs (62.5 µg/mL for E. coli) or H-AgNPs (125 µg/mL for S. aureus) alone. Their high combined inhibitory effect against E. coli (NF-1-3) was synergistic and against S. aureus (NF-2 and NF-3) potentially additive. Considering such promising results, we believe our study provides some direction for new research and strategies in antimicrobial therapeutics.
Srebrne se nanočestice (AgNP), koje su već neko vrijeme u središtu pažnje zbog svojih antimikrobnih svojstava, mogu proizvoditi i zelenom sintezom. Cilj je ovog istraživanja bio (i) opisati (karakterizirati) zelenu sintezu različitih AgNP-a pomoću vodenih ekstrakata čekinjaste broćike (Galium aparine) (G-AgNPs) i pješčarskoga smilja (Helichrysum arenarium) (H-AgNPs), dobivenih metodom mikrovalne ekstrakcije, te (ii) utvrditi antimikrobno djelovanje kombinacije tih dvaju nanosustava u različitim omjerima. Oblikovanje nanočestica i kemijske reakcije utvrđene su pomoću UV-Vis spektroskopije. Veličina G-AgNP-a bila je 52,0±10,9 nm, njihov polidisperzivni indeks (PDI) 0,285±0,034, a zeta potencijal -17,9±0,9 mV. Osobine H-AgNP-a bile su sljedeće: veličina 23,9±1,0 nm, PDI 0,280±0,032, a zeta potencijal -21,3±2,7 mV. Mikroskopijom atomskih sila (engl. atomic force microscopy, krat. AFM) i pretražnom elektronskom mikroskopijom (engl. scanning electron microscopy, krat. SEM) potvrđeno je da su čestice monodisperzivne i sferične. Rezultati infracrvene spektroskopije s Fourierovom transformacijom (engl. Fourier transform-infrared spectroscopy, krat. FT-IR) potvrdili su prisutnost reduktivnih agenasa koji su stabilizirali srebrne nanočestice. Zatim su pripremljene tri formulacije nanočestica (NF-1, NF-2 i NF-3) kombinacijom sintetiziranih nanočestica u različitim omjerima, a njihova antimikrobna djelotvornost testirana je na mikroorganizmima E. coli, S. aureus, C. albicans i A. flavus. Naše je istraživanje prvo koje dokazuje da kombinacija srebrnih nanočestica dobivenih iz dvaju bioloških izvora može biti djelotvorna te da ima poboljšano antibakterijsko djelovanje protiv E. coli i S. aureus u odnosu na zasebne nanosustave. Minimalna inhibicijska koncentracija kombinacija iznosila je 31,25 µg/mL za E. coli u svim nanoformulacijama te 62,5 µg/mL za S. aureus s NF-1, odnosno 125 µg/mL s NF-2 i NF-3, a minimalne inhibicijske koncentracije G-AgNP-a odnosno H-AgNP-a zasebno su iznosile 62,5 µg/mL za E. coli (G-AgNP), odnosno 125 µg/mL za S. aureus (H-AgNP). To kombinirano antibakterijsko djelovanje protiv E. coli bilo je sinergijsko, a protiv S. aureus naizgled aditivno. S obzirom na ovako obećavajuće rezultate, smatramo da naše istraživanje daje smjer za razvoj novih strategija u antibakterijskom liječenju.

Aspergillus favus (A. flavus) is a saprophytic fungus and a pathogen affecting several important foods and crops, including maize. A. flavus produces a toxic secondary metabolite called aflatoxin. Alpha-amylase (α-amylase), a hydrolytic enzyme produced by A. Flavus helps in the production of aflatoxin by hydrolysing the starch molecules in to simple sugars such as glucose and maltose. These simple sugars induce the production of aflatoxin. Inhibition of α-amylase has been proven as a potential way to reduce the production of aflatoxin. In the present study, we investigated the effect of selected carboxylic acid derivatives such as cinnamic acid (CA), 2, 4-dichlorophenoxyacetic acid (2,4-D), and 3-(4-hydroxyphenyl)-propionic acid (3,4-HPPA) on the fungal growth and for the α-amylase inhibitory activity. The binding potentials of these compounds with α-amylase have been confirmed by enzyme kinetics and isothermal titration calorimetry. Molecular docking and MD simulation studies were also performed to deduce the atomic level interaction between the protein and selected ligands. The results indicated that CA, 2,4-D and 3,4-HPPA can inhibit the fungal growth which could be partly due to the inhibition on fungal α-amylase activity.Communicated by Ramaswamy H. Sarma.

Aspergillus flavus and Aspergillus fumigatus are important human pathogens that can infect the lung and cornea. During infection, Aspergillus dormant conidia are the primary morphotype that comes in contact with the host. As the conidial surface-associated proteins (CSPs) and the extracellular proteins during the early stages of growth play a crucial role in establishing infection, we profiled and compared these proteins between a clinical strain of A. flavus and a clinical strain of A. fumigatus. We identified nearly 100 CSPs in both Aspergillus, and these non-covalently associated surface proteins were able to stimulate the neutrophils to secrete interleukin IL-8. Mass spectrometry analysis identified more than 200 proteins in the extracellular space during the early stages of conidial growth and germination (early exoproteome). The conidial surface proteins and the early exoproteome of A. fumigatus were enriched with immunoreactive proteins and those with pathogenicity-related functions while that of the A. flavus were primarily enzymes involved in cell wall reorganization and binding. Comparative proteome analysis of the CSPs and the early exoproteome between A. flavus and A. fumigatus enabled the identification of a common core proteome and potential species-specific signature proteins. Transcript analysis of selected proteins indicate that the transcript-protein level correlation does not exist for all proteins and might depend on factors such as membrane-anchor signals and protein half-life. The probable signature proteins of A. flavus and A. fumigatus identified in this study can serve as potential candidates for developing species-specific diagnostic tests. KEY POINTS: • CSPs and exoproteins could differentiate A. flavus and A. fumigatus. • A. fumigatus conidial surface harbored more antigenic proteins than A. flavus. • Identified species-specific signature proteins of A. flavus and A. fumigatus.