Aflatoxigenic Strains Research Articles

Impact of simulated climate change conditions on Aspergillus flavus biocontrol effectiveness in peanut-based medium and peanut seeds

Peanut is a legume widespread in the world, but its high susceptibility to Aspergillus flavus infection poses a significant challenge due to the risk of aflatoxin contamination. It is predicted that changing climatic conditions will result in warmer, drier periods with elevated CO₂ levels, which promote the growth of A. flavus. The most effective pre-harvest mitigation strategy is the use of non-aflatoxigenic strains of biocontrol; however, future climatic conditions may influence the effectiveness of this practice. Thus, the objective of this study was to assess the impact of simulated climate change conditions on the efficacy of a non-aflatoxigenic A. flavus strain, the active agent of a biocontrol product, in reducing fungal growth and mycotoxin production. A range of temperature conditions (T = 25, 30, 35 °C), water activity (aw = 0.85, 0.90, 0.95) and CO2 concentration (400, 1000 ppm) were selected for investigation. The assay was conducted using three ratios of A. flavus spore suspensions (100 % aflatoxigenic, 100 % non-aflatoxigenic, 50/50 % aflatoxigenic:non-aflatoxigenic; 105 spores mL−1) inoculated in vitro on a peanut-based medium (PBM), and in situ on peanut seeds, and incubated for 10 days. Results of in vitro studies showed a significant influence of T and aw on fungal growth rates (μ), with a reduction when the aw decreased and T shifted from the fungus's optimum of 30 °C. The highest mycotoxin concentration was detected on PBM, with an aflatoxin B1 (AFB1) production by the aflatoxigenic strain in situ 50 % lower than in vitro. However, for all the treatments, the application of the biocontrol agent inhibited AFB1 production with a general reduction of 55 % in vitro and 71 % in situ, even though a significant increase in kojic acid production was observed. The effectiveness of the non-aflatoxigenic strain increased when T was raised up to 35 °C with higher AFB1 reductions both in vitro and in situ, of respectively 58 and 76 %.These observations provided the first evidence that climate change will not negatively influence the ability of the Italian A. flavus non-aflatoxigenic strain, which represents the biocontrol agent of the commercial product AF-X1, to reduce AFB1 contamination in peanuts.

Biocontrol of Aflatoxin-Producing Aspergillus flavus ATCC 22546 by a Non-Aflatoxigenic Aspergillus flavus ATCC 9643

The biological control of Aspergillus flavus and A. parasiticus by non-aflatoxigenic strains has been introduced in cotton- and peanut-cultivating fields and proven successful at reducing aflatoxin (AFB) contamination of crops and agricultural soils. In this study, a non-aflatoxigenic strain, A. flavus ATCC 9643 (ATCC 9643), was evaluated for its ability to competitively inhibit the growth of an aflatoxigenic strain, A. flavus ATCC 22546 (ATCC 22546), and mitigate AFB production in ATCC 22546 during competitive growth. To comparatively analyze the suppressive effect of ATCC 9643 on ATCC 22546, a non-aflatoxigenic strain (A. flavus ATCC 96045, known as AF36) was used as a positive control in some experiments. The two non-aflatoxigenic strains did not produce AFB1 or AFB2 owing to the absence of several AFB biosynthesis-related genes, especially aflK and aflL, which encode versicolorin B synthase and desaturase, respectively. To create a competitive growth environment, ATCC 9643 and ATCC 22546 were co-inoculated into a solid agar medium, and they grew at similar rates when added at a 1:1 ratio. Increasing the inoculum rate of ATCC 9643 (1:1, 1:3, 1:5) dramatically inhibited ATCC 22546 growth, and AFB production was effectively decreased by about 84%, 95%, and 97% by treatment with ATCC 9643. On rice, ATCC 22546 attenuated ATCC 9643 growth only when the rice was submerged in distilled water, whereas agar addition enhanced it. Taken together, ATCC 9643 is a promising candidate biological agent for suppressing aflatoxigenic A. flavus strain growth and alleviating AFB contamination. Further studies on AFB reduction in crop fields, including cotton-cultivation and maize-cultivation fields, are warranted.

Three Ecological Models to Evaluate the Effectiveness of Trichoderma spp. for Suppressing Aflatoxigenic Aspergillus flavus and Aspergillus parasiticus.

Chemical pesticides help reduce crop loss during production and storage. However, the carbon footprints and ecological costs associated with this strategy are unsustainable. Here, we used three in vitro models to characterize how different Trichoderma species interact with two aflatoxin producers, Aspergillus flavus and Aspergillus parasiticus, to help develop a climate-resilient biological control strategy against aflatoxigenic Aspergillus species. The growth rate of Trichoderma species is a critical factor in suppressing aflatoxigenic strains via physical interactions. The dual plate assay suggests that Trichoderma mainly suppresses A. flavus via antibiosis, whereas the suppression of A. parasiticus occurs through mycoparasitism. Volatile organic compounds (VOCs) produced by Trichoderma inhibited the growth of A. parasiticus (34.6 ± 3.3%) and A. flavus (20.9 ± 1.6%). The VOCs released by T. asperellum BTU and T. harzianum OSK-34 were most effective in suppressing A. flavus growth. Metabolites secreted by T. asperellum OSK-38, T. asperellum BTU, T. virens OSK-13, and T. virens OSK-36 reduced the growth of both aflatoxigenic species. Overall, T. asperellum BTU was the most effective at suppressing the growth and aflatoxin B1 production of both species across all models. This work will guide efforts to screen for effective biological control agents to mitigate aflatoxin accumulation.

Evaluation of Antifungal and anti-aflatoxin B1 efficacy of some crude extracts of Chamaerops humilis L. against Aspergillus flavus isolated from peanuts (Arachis hypogea L.)

The present study strengthens the food preservative potential of Chamaerops humilis extracts viz. hexane extract, chloroform extract, ethanol extract, chloroform extract, and methanol extract based on their antifungal, antiaflatoxin, and antioxidant efficacy. The isolation of molds from peanuts were carried out by the suspension-dilution technique and inoculation on agar medium. Antifungal determinations were conducted using the agar plate and liquid dilution methods. The effect of C. humilis L. extracts on the production of AFB1 was determined in a SMKY broth. The molds isolated were of the genera Aspergillus, Penicillium sp., Fusarium sp., and Alternaria sp. with A. flavus (BBH-6) which is identified as the highest AFB1 producer. The minimum inhibitory concentration of extracts against the toxigenic strain of A. flavus ranged between 3.00 mg/ml and 3.50 mg/ml. The extracts were found more efficacious as they inhibited the dry mycelium weight and aflatoxin production of the aflatoxigenic strain A. flavus (BBH-6) at lower concentrations. The extracts showed fungitoxic spectrum against four molds. The IC50 value of C. humilis L. ranged between 140.4 and 189.46 μg/mg, oxidation of linoleic acid was moderately inhibited by the extracts ranges between 53.54 and 69.33%, while their total phenolic content ranged between 47.80 and 115.71 μg/ml. Based on the findings of present investigation, C. humilis L. can be introduced as a proposed solution for formulating plant-based preservative food additives.

Identification and biodiversity patterns of Aspergillus species isolated from some soil invertebrates at high altitude using morphological characteristics and phylogenetic analyses.

The carcinogenic, mutagenic, and teratogenic chemicals such as aflatoxin are a worldwide health problem. Aspergillus spp., responsible for most cases of aflatoxin contamination, are common in the environment and spread easily to many different types of food. The objectives of this study were to conduct a survey of fungi associated with three soil invertebrates in Taif, Saudi Arabia, identify these isolates and explore mycotoxins formation. In total, 114 fungal isolates were collected from various soil invertebrates (millipedes, Armadillidium vulgare and Porcellio laevis) in Taif, Saudi Arabia, among them, 22 isolates were identified as Aspergillus spp. based on morphological and molecular characteristics followed by both Fusarium and Penicillium. The sequences of ITS 1 and ITS 4 were utilized. Using bootstrap analysis, phylogenetic tree was split into two distinct clusters. Five sub clusters were included inside the first major cluster, and their bootstrap value was 99%. While, there were two small clusters in the second major cluster. All the tested Aspergillus strains were able to have a single PCR fragment amplified using the primer AspTef. TEF-1 DNA sequence bootstrap analysis with 1,000 replicates revealed two distinct groups. Additionally, the Aspergillus isolates were grouped into two different clusters with about 65% genetic similarity using ISSR-PCR analysis. The standard polymerase chain reaction was used to effectively amplify the Aopks, afl-A and omt-A genes in aflatoxigenic Aspergillus strains. Four Aspergillus strains used in this investigation were shown to generate aflatoxin B1. While, three Aspergillus stains showed ochratoxin genes. In conclusion, the results indicate significant differences in the fungal community between ecoregions and soil invertebrates. Moreover, mycotoxin detection and identification among Aspergillus isolates were elucidated. This study could shed light on the risk of mycotoxin contamination along the supply chain.

Molecular characterization of aflatoxigenic and non-aflatoxigenic Aspergillus flavus strains isolated from peanut sold in northern Togo

The contamination of agricultural products and aflatoxins synthesis can be controlled and reduced by the application of a biological control product based on atoxinogenic Aspergillus flavus strains. The purpose of this work was to select atoxinogenic Aspergillus flavus strains that can be used in the biological control of aflatoxins. Thirty-four (34) strains of Aspergillus flavus were used and the Nor-1, Ver-1, Omt-A and AflR genes involved in the aflatoxin biosynthesis pathway were sought. The growth speed and ability of atoxigenic strains to inhibit the growth of toxigenic strains were also determined. The results showed that 50% of the strains have at least one of the genes sought and the percentages of inhibition (PI) of the atoxinogenic strains varied significantly from 16.98% to 62.50% at the threshold of 5% according to the Tukey test (p-value = 0.0000136). The mycelial growth speed (VCM) of the atoxinogenic strains varied from 7.5 to 10 mm/day with an average VCM of 8.4 ± 0.05 mm/day. Strains AKA-10; AGA-8; AMAN-35; ABA-28 and AGA-46 were found to be more effective in inhibiting mycelial growth of toxigenic strains.

Detoxification of Aflatoxins in Fermented Cereal Gruel (Ogi) by Probiotic Lactic Acid Bacteria and Yeasts with Differences in Amino Acid Profiles

Toxigenic members of Aspergillus flavus contaminate cereal grains, resulting in contamination by aflatoxin, a food safety hazard that causes hepatocellular carcinoma. This study identified probiotic strains as aflatoxin detoxifiers and investigated the changes to the grain amino acid concentrations during fermentation with probiotics in the presence of either A. flavus La 3228 (an aflatoxigenic strain) or A. flavus La 3279 (an atoxigenic strain). Generally, higher concentrations (p < 0.05) of amino acids were detected in the presence of toxigenic A. flavus La 3228 compared to the atoxigenic A. flavus La 3279. Compared to the control, 13/17 amino acids had elevated (p < 0.05) concentrations in the presence of the toxigenic A. flavus compared to the control, whereas in systems with the atoxigenic A. flavus 13/17 amino acids had similar (p > 0.05) concentrations to the control. There were interspecies and intraspecies differences in specific amino acid elevations or reductions among selected LAB and yeasts, respectively. Aflatoxins B1 and B2 were detoxified by Limosilactobacillus fermentum W310 (86% and 75%, respectively), Lactiplantibacillus plantarum M26 (62% and 63%, respectively), Candida tropicalis MY115 (60% and 77%, respectively), and Candida tropicalis YY25, (60% and 31%, respectively). Probiotics were useful detoxifiers; however, the extent of decontamination was species- and strain-dependent. Higher deviations in amino acid concentrations in the presence of toxigenic La 3228 compared to atoxigenic La 3279 suggests that the detoxifiers did not act by decreasing the metabolic activity of the toxigenic strain.

土壌サンプル中のアフラトキシン産生菌の生残を調査するための寒天培地全量抽出法

In this study, we investigated a method of extracting the whole agar dish culture to assess the existence and survival of producing fungi of aflatoxins (AFs) in field soil samples. In a control experiment using a known aflatoxigenic Aspergillus flavus strain with dichlorvos (DV) - coated agar or DV-free agar, no significant difference on the recovery of colony numbers was observed while DV clearly inhibited AFs accumulation. Therefore, a method of extracting the whole dish culture of DV-free agar followed by aflatoxin B1 (AFB1) analysis was applied to assess the survival of aflatoxigenic fungi in soil samples during storage. Soil samples stored at different temperature (4°C, -20°C, and -80°C) and duration (3-12 months) were cultured and analyzed for the accumulation of AFB1 in triplicate. Each soil sample suspension was cultured on DV-free agar for 7 days at 25°C, and the whole dish culture was extracted by methanol and the amount of AFB1 was measured. AFB1 amounts were drastically decreased after the fourth month of storage at all temperatures tested. After the fifth month, AFB1 amounts of samples stored at 4°C were significantly lower than those stored at -20°C or -80°C, indicating that temperatures below -20°C may be suitable for longer storage of soil samples. This whole dish culture extraction method is easy to handle and will be applicable to estimate the population of aflatoxigenic fungi among various soil samples from different origin or conditions.

Mutual effects on mycotoxin production during co-culture of ochratoxigenic and aflatoxigenic Aspergillus strains.

Mycotoxin co-occurrence compromises the safety of food crops worldwide. Environmental factors, as well as fungal interaction, can substantially influence the infectivity of mycotoxigenic fungi and their subsequent production of multi-mycotoxin. Here, we investigated the mutual effects of the co-culture of ochratoxigenic and aflatoxigenic Aspergillus strains on the co-production of ochratoxin A (OTA) and aflatoxin B1 (AFB1). Single cultures of ochratoxigenic A. carbonarius and A. alliaceus grew optimally at 25°C, whereas aflatoxigenic A. flavus grew optimally at 35°C. The maximum levels of OTA and AFB1 were achieved at 25°C, whereas mycotoxin production decreased at 35°C. During competitive growth of the ochratoxigenic and aflatoxigenic isolates, inhibition or stimulation of mycotoxin production was dependent on the fungal strain, temperature, and the ratio of the spore concentration. Aspergillus carbonarius and A. alliaceus generally produced OTA, with similar patterns of relative OTA levels at all temperatures. AFB1 production by A. flavus in the presence of ochratoxigenic Aspergillus species was inhibited at 25°C and stimulated at 35°C. These results indicated that the temperature, presence of other mycotoxigenic Aspergillus species, and ratio of the initial spore concentration significantly contributed to the co-production of OTA and AFB1.

Antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate ofStreptomyces philanthiRL-1-178 on the two aflatoxigenic fungi and identification of its active component

The study reports the antifungal and antiaflatoxigenic mechanism activity of freeze-dried culture filtrate of Streptomyces philanthi RL-1-178 (DCF RL-1-178) against two aflatoxigenic strains (Aspergillus parasiticus and A. flavus) and identification of its active component. Significant inhibition in ergosterol biosynthesis by the DCF RL-1-178 appeared on the plasma membrane. Moreover, the DCF RL-1-178 showed dose-dependent inhibition of methylglyoxal (MG) (an aflatoxin inducer) biosynthesis and exhibited a novel antiaflatoxigenic action mechanism. Significant impairments in enzymatic [superoxide dismutase (SOD) and catalase (CAT)] and nonenzymatic [oxidized and reduced glutathione (GSH) and ratio of oxidized and reduced glutathione (GSSG)] anti-oxidative defense molecules were observed in the two aflatoxigenic cells. The active component of the DCF RL-1-178 was identified as natamycin. The natamycin exhibited against A. parasiticus and A. flavus with the minimum inhibitory concentration (MIC) values of 0.5 and 1.0 µg ml-1, respectively, while the minimum fungicidal concentration values were the same (4.0 µg ml-1). The DCF RL-1-178 containing natamycin exhibited the following effects: (1) inhibition of cellular ergosterol biosynthesis on plasma membrane, (2) reduction in MG (aflatoxin inducer) confirmed novel antiaflatoxigenic mechanism of action, and (3) caused remarkable debasement in antioxidant defense enzymes (SOD and CAT) and nonenzymatic defense molecules (GSH and GSSG) revealing biochemical mechanism of action.

Evaluation of Aflatoxin-Producing Fungi in Indoor Air of Warehouses and Houses of Farmers in Giwa, Kaduna State Nigeria

The health risks associated with ingesting food contaminated with mycotoxins, particularly aflatoxin-contaminated staple foods like maize and other cereals, have been widely studied. However, there is little knowledge about the role of inhalation of pathogenic fungi as bioaerosols in contaminated air from handling crops as an occupational health risk. This paper presents a study aimed at determining the level of airborne aflatoxin-producing fungi in the indoor air of grain stores in the Giwa community of Kaduna State. Indoor air was sampled using the settling plate technique from grain stores, warehouses and living rooms. Metrological data of the studied area were collected from the Institute of Agricultural Research, ABU, Zaria. Airborne mycofloral concentrations were determined, and colonies of Aspergillus flavus were identified. The isolates were screened for aflatoxin production on Neutral Red Desiccated Coconut Agar (NRDCA). Selected aflatoxin-producing fungal isolates were screened for the presence of aflD (nor-1), aflM (ver-1) and AflR genes by PCR. Sampling was done once every month from October to December 2020. Mycofloral concentrations were in the range of 2.77x103−4.05x103 and 1.55x103−2.17x103CFUm-3for grain stores and living rooms respectively. A total of twelve (12) strains of A. flavus were isolated from the indoor air of the grain stores and warehouses while none was obtained from the living room. Eleven (11) isolates were confirmed to be aflatoxigenic on NRDCA, presenting 30 CFUm-3 of the indoor air mycofloral composition. The aflD, aflM and aflR were amplified with aflD being the most detected gene from all the aflatoxin-producing mould isolates of Aspergillus species. The mycofloral concentrations in the grain stores were higher than those in the living room and, in all the sampling sites, exceeded the limit of the total mycofloral concentration of 500 CFUm-3 for agricultural and industrial environments. There were significant differences (p&lt;0.05) in the indoor mycofloral concentrations between the grain stores/warehouses and the living room. The presence of aflatoxigenic strains of A. flavus in the stores indicates that grain handlers and traders are at risk of occupational exposure to aflatoxigenic fungi and aflatoxins. Hence, they should wear protective materials for their safety while working in such stores.

Insights on biorational potential of Ocimum gratissimum essential oil and its binary combination with monoterpene phenol for control of rice weevil (Sitophilus oryzae) and aflatoxigenic fungi.

Food grain storage is a difficult task due to insect infestation and subsequent mycotoxin contaminations which adversely affects the nutritional quality of grains and leading to economic loss. Current research focuses on contact and fumigant toxicity effects of essential oils (EO) and aroma compounds against Sitophilus oryzae and growth inhibition of aflatoxigenic fungi. The EO of Ocimum gratissimum comprised of thymol (46.8%), γ-terpinene (14.04%) along with o-cymene (11.76%). Also, Cymbopogon flexuosus rich in citral (76.3%) and geraniol (84.6%) and Cymbopogon nardus having geraniol (49.24%) and geranyl acetate (20.9%) were all evaluated using a dose of 25 μL (Conc∼833.3 μL/L air) against S. oryzae. All the compounds showed significant mortality (>95%) at 24 h of exposure. The insecticidal property of O. gratissimum oil chemotype showed a strong contact and fumigant toxicity against S. oryzae at a highest dose of 25 μL (Conc∼833.3 μL/L air) within 24 h. It has been further evaluated on three aflatoxigenic fungal strains which showed reduction in growth and aflatoxin content (Aflatoxin B and G), which was markedly reduced upon the treatment. The binary mixture interaction of O. gratissimum oil with monoterpene phenol (carvacrol) was assessed and the specific binary mixture of 80:20 ratio (having additive property) acts as a contact insecticide with 100% mortality. Hence, essential oil of less explored Ocimum species (O. gratissimum) and its binary mixture could be deployed as potential biorational for control of rice weevil (Sitophilus oryzae) and aflatoxigenic Aspergillus spp.

Preparation and characterization of a novel nanoemulsion consisting of chitosan and Cinnamomum tamala essential oil and its effect on shelf-life lengthening of stored millets

This study aimed to improve the stability of Cinnamomum tamala essential oil (CTEO) via encapsulating into chitosan nanoemulsion (CsNe) through an ionic-gelation technique and explore its food preservative efficacy against aflatoxigenic strain of Aspergillus flavus (AFLHPSi-1, isolated from stored millet), aflatoxin B1 (AFB1) contamination, and lipid peroxidation, causing qualitative deterioration of stored millets. The CTEO was characterized through gas chromatography–mass spectrometry (GC–MS) analysis that confirmed the presence of linalool as a major component occupying approximately 82.64% of the total oil. The synthesized nanoparticles were characterized through scanning electron microscopy (SEM), fourier transform infrared (FTIR) spectroscopy, and X-ray diffraction (XRD) analysis. The encapsulation efficiency (EE) and loading capacity (LC) of CTEO-CsNe were found to be 97.71% and 3.33%, respectively. In vitro release study showed a biphasic release pattern: with an initial burst release followed by a controlled release of CTEO. During investigation of efficacy, the CTEO-CsNe caused complete inhibition of A. flavus growth, and AFB1 biosynthesis at 1.0 and 0.8 μL/mL, respectively. The CTEO-CsNe exhibited its antifungal mode of action by altering fungal plasma membrane integrity (ergosterol inhibition) and permeability (leakage of important cellular constituents), and antiaflatoxigenic mode of action by inhibiting cellular methylglyoxal biosynthesis. CTEO-CsNe showed high free radical scavenging capacity (IC50 = 5.08 and 2.56 μL/mL) against DPPH•+ and ABTS•+ radicals, respectively. In addition, CTEO-CsNe presented remarkable preservative efficacy, inhibiting AFB1 and lipid peroxidation in model food system (Setaria italica) without altering their organoleptic properties. Based on overall results, CTEO-CsNe can be recommended as a novel shelf-life enhancer of stored millet samples.

Contamination of Stored Pistachios to Aspergillus Section Flavi and Aflatoxins

Background: Contamination of stored pistachio nuts to Aspergillus section Flavi and aflatoxins (AFs) as well as effective parameters on fungal growth and AF production during storage was assayed.&#x0D; Methods: Twenty one pistachio nut samples were taken from some warehouse of Damghan county. Different parameters of storage, including temperature, relative humidity, and pest infestation were recorded. Contamination to aflatoxigenic fungi was investigated by serial dilution method and aspergillus flavus and parasiticus agar (AFPA) medium with three repetitions. Thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) method were used to measure quality and quantity of the produced AFs, respectively.&#x0D; Results: There was a significant difference in contamination to Aspergillus section Flavi between different samples on the basis of statistical analysis. There was no significant difference between samples contamination to Aspergillus section Flavi and storage parameters. The ability of AF production of isolates was variable. More than 88% of isolates were able to produce one or several types of AFs and less than 12% of isolates produced no AF. HPLC assay showed that out of 21 pistachio nut samples, 3 samples were contaminated with various types of AFs, but were below the allowable levels (P ≤ 0.05).&#x0D; Conclusion: Despite the population of aflatoxigenic strains in the mycoflora of warehouse, AF contamination of pistachios was estimated to be about 14%, which was also less than the allowable level. The results showed that the environmental parameters required for the production of AF in the warehouse of Semnan province do not exist and the storage conditions in this province are suitable.

Assessment of the Potential of a Native Non-Aflatoxigenic Aspergillus flavus Isolate to Reduce Aflatoxin Contamination in Dairy Feed.

Aspergillus species can produce aflatoxins (AFs), which can severely affect human and animal health. The objective was to evaluate the efficacy of reducing AF contamination of a non-aflatoxigenic isolate of A. flavus experimentally coinoculated with different aflatoxigenic strains in whole plant (WP), corn silage (CS), immature grains (IG) and in culture media (CM). An L-morphotype of A. flavus (CS1) was obtained from CS in a dairy farm located in the Mexican Highland Plateau; The CS1 failed to amplify the AFs biosynthetic pathway regulatory gene (aflR). Monosporic CS1 isolates were coinoculated in WP, CS, IG and CM, together with A. flavus strains with known aflatoxigenic capacity (originating from Cuautitlán and Tamaulipas, Mexico), and native isolates from concentrate feed (CF1, CF2 and CF3) and CS (CS2, CS3). AF production was evaluated by HPLC and fungal growth rate was measured on culture media. The positive control strains and those isolated from CF produced a large average amount of AFs (15,622 ± 3952 and 12,189 ± 3311 µg/kg), whereas A. flavus strains obtained from CS produced a lower AF concentration (126 ± 25.9 µg/kg). CS1 was efficient (p < 0.01) in decreasing AF concentrations when coinoculated together with CF, CS and aflatoxigenic positive control strains (71.6–88.7, 51.0–51.1 and 63.1–71.5%) on WP, CS, IG and CM substrates (73.9–78.2, 65.1–73.7, 63.8–68.4 and 57.4–67.6%). The results suggest that the non-aflatoxigenic isolate can be an effective tool to reduce AF contamination in feed and to minimize the presence of its metabolites in raw milk and dairy products intended for human nutrition.

Medium for the Production of Bacillus-Based Biocontrol Agent Effective against Aflatoxigenic Aspergillus flavus: Dual Approach for Modelling and Optimization.

One of the leading limiting factors for wider industrial production and commercialization of microbial biopesticides refers to the high costs of cultivation media. The selection of alternative sources of macronutrients crucial for the growth and metabolic activity of the producing microorganism is a necessary phase of the bioprocess development. Gaining a better understanding of the influence of the medium composition on the biotechnological production of biocontrol agents is enabled through bioprocess modelling and optimization. In the present study, after the selection of optimal carbon and nitrogen sources, two modelling approaches were applied to mathematically describe the behavior of the examined bioprocess—the production of biocontrol agents effective against aflatoxigenic Aspergillus flavus strains. The modelling was performed using four independent variables: cellulose, urea, ammonium sulfate and dipotassium phosphate, and the selected response was the inhibition-zone diameter. After the comparison of the results generated by the Response Surface Methodology (RSM) and the Artificial Neural Network (ANN) approach, the first model was chosen for the further optimization step due to the better fit of the experimental results. As the final investigation step, the optimal cultivation medium composition was defined (g/L): cellulose 5.0, ammonium sulfate 3.77, dipotassium phosphate 0.3, magnesium sulfate heptahydrate 0.3.

Unveiling the cellular and molecular mode of action of Melaleuca cajuputi Powell. essential oil against aflatoxigenic strains of Aspergillus flavus isolated from stored maize samples

This study aimed to reveal the bio-efficacy of Melaleuca cajuputi essential oil (McEO) against aflatoxigenic fungi and lipid peroxidation causing deterioration of stored maize samples. Three different toxigenic strains of Aspergillus flavus, namely AF-LHP-M2, AF-LHP-SP2, and AF-LHP-VS8 were investigated. Gas chromatography-mass spectrometry (GC-MS) analysis of EO revealed the presence of α-pinene (49.24%) as major compound. Investigation on efficacy showed that McEO exhibited remarkable inhibitory activity against growth and AFB1 production by AF-LHP-M2 (2.0 and 1.4 μL mL−1, respectively), AF-LHP-SP2 (1.2 and 1.0 μL mL−1, respectively), and AF-LHP-VS8 (0.8 μL mL−1) (p < 0.05). The McEO inhibited fungal growth via inhibition of ergosterol biosynthesis, cellular constituents' leakage, and damage of mitochondrial membrane potential, while AFB1 production by inhibition of intracellular methylglyoxal. Further, molecular docking study was carried out to unveil the binding affinities of major compounds with the target protein Nor-1 (primarily catalyze an important step in AFB1 biosynthesis), and the results revealed good correlation with the experimental findings. In addition, McEO showed significantly (p < 0.05) higher DPPH• and ABTS•+ scavenging activity with IC50 values 3.16 and 4.29 μL mL−1, respectively. Interestingly, McEO inhibited AFB1 production, and malondialdehyde content in fumigated maize samples without significantly (p < 0.05) changing their sensory attributes, ascertaining its efficacy in food system with high safety profile (LD50 = 1800 mg kg−1 body weight) on mice model. The overall results proved McEO's potential as natural food preservative of stored food products.

Detection of Aspergillus flavus in Wheat Grains Using Anti-mannoprotein (MP1) and Spore Protein Polyclonal Antibodies

Cell wall mannoprotein (MP1) gene of an aflatoxigenic strain of Aspergillus flavus, isolated from stored wheat grains, was cloned and sequenced. MP1 protein was expressed in E. coli in soluble form and purified. Polyclonal antibodies were raised against recombinant MP1 protein and inactivated spores of this fungus in rabbit and purified by ammonium sulphate precipitation, Protein A sepharose and antigen affinity chromatography. The minimum concentration of purified mycelial or spore proteins that could be detected by ELISA was determined as 100ng using 2µg of these antibodies. The anti-MP1 antibody was found more sensitive than anti-spore protein antibody. Western blot and immunofluorescence analysis showed reactivity of these antibodies to various proteins (30 to 200kDa) distributed throughout the surface of mycelia and spore of A. flavus. Cross-reactivity of these antibodies was detected with fungi belonging to different Aspergillus, Rhizopus and Alternaria species out of fourteen different fungal species tested. In fungal contaminated wheat grains, these antibodies could detect presence of as low as 1µg mycelia or 103 spores per gram of wheat grains using ELISA. The results suggest that the developed antibodies could be successfully applied for the detection of predominant fungal infestation in stored wheat grains.

Incidence of mycobiota and aflatoxin B1 in Algerian feed

The presence of fungi and aflatoxin B1 (AFB1) in 101 animal feed samples randomly collected from different vendors and factories in Algeria was investigated. For fungi, the main genera isolated were Aspergillus, Penicillium and Fusarium. Furthermore, the 459 strains of Aspergillus section Flavi were screened for their ability to produce aflatoxins and cyclopiazonic acid (CPA). 49% of the strains produced AFB1 while 74.5% were cyclopiazonic acid producers. The highest incidence of aflatoxigenic strains was recorded in maize (61%) and ground poultry feed (60%). The presence of AFB1 in 101 feed samples was evaluated using HPLC-FLD. The obtained data showed that 36.6% of samples were contaminated in the range of 0.34 to 171.06 μg/kg. Six samples exceeded the Algerian maximum limit of 20 μg/kg for AFB1. This study highlights the potential presence of aflatoxigenic strains belonging to section Flavi and AFB1 in animal feed in Algeria, strategic information for the Algerian policymakers.

Analysis of the competitiveness between a non-aflatoxigenic and an aflatoxigenic Aspergillus flavus strain on maize kernels by droplet digital PCR

Non-aflatoxigenic Aspergillus flavus strains are used as a biocontrol system on maize fields to decrease the aflatoxin biosynthesis of aflatoxigenic A. flavus strains. A. flavus strain AF36 was the first commercially available biocontrol strain and is authorized for use on maize fields by the US Environmental Protection Agency, e.g., in Texas and Arizona. A droplet digital PCR (ddPCR) assay was developed to analyze the mechanisms of competition and interaction of aflatoxigenic and non-aflatoxigenic A. flavus strains. This assay enables the parallel identification and quantification of the biocontrol strain A. flavus AF36 and the aflatoxigenic A. flavus strain MRI19. To test the assay, spores of both strains were mixed in varying ratios and were incubated on maize-based agar or maize kernels for up to 20 days. Genomic equivalent ratios (genome copy numbers) of both strains were determined by ddPCR at certain times after incubation and were compared to the spore ratios used for inoculation. The aflatoxin biosynthesis was also measured. In general, A. flavus MRI19 had higher competitiveness in the tested habitats compared to the non-aflatoxigenic strain, as indicated by higher final genomic equivalent ratios of this strain compared to the spore ratios used for inoculation. Nevertheless, A. flavus AF36 effectively controlled aflatoxin biosynthesis of A. flavus MRI19, as a clear aflatoxin inhibition was already seen by the inoculation of 10% spores of the biocontrol strain mixed with 90% spores of the aflatoxigenic strain compared to samples inoculated with only spores of the aflatoxigenic A. flavus MRI19.