Aflatoxigenic Strains Research Articles

An in Vitro Antifungal and Antiaflatoxigenic Properties of Commiphora myrrha and Prunus mahaleb

Aflatoxins and especially aflatoxin B, are the devastating contaminant of food and feed products with hazardous effects to mankind and his domestic animals. These investigations were set to evaluate the effect of various levels of Commiphora myrrha resin (1.0, 1.25, 2.25, and 3.25 g/100 ml) and Prunus mahaleb seed extract (0.75, 1.5, 2.5, and 3.5 g/100 ml) on the growth and aflatoxin secretion by two aflatoxigenic strains of Aspergillus flavus and A. parasiticus. The two plant extracts significantly (p<0.05) decreased aflatoxin secretion, and inhibited the fungal growth. Resin of C. myrrha displayed 51.9-95.7% reduction in total aflatoxin secretion by A. flavus, and 46.9-92% for A. parasiticus, and Seed extract of P. mahaleb decreased aflatoxin up to 53.7-95.8% and 40-94.7%, respectively. The inhibition of aflatoxin B (B1 and B2) by myrrh resin and seed extract of mahaleb ranged between 51.7-93.5, 50-93.6% (A. flavus) and 39.5-89.7%, 37.9-93% (A. parasiticus). The mycelial dry weight of A. flavus and A. parasiticus ws decreased up to 46.1-58.7%, 28.9-51.3% (Myrrh resin), and between 45-56.9%, 33.3-55.9% (Mahaleb seed extract). Nonetheless, the two plant extracts did not detoxify aflatoxin B1. Therefore, it apparent that the resin of C. myrrha and seed extract of P. mahaleb affected the biosynthesis pathway of aflatoxins. Thus, they can be recommended as effective natural plant biopreservative against aflatoxin contamination of food and feed products.

Combinational inhibitory action of essential oils and gamma irradiation for controlling Aspergillus flavus and Aspergillus parasiticus growth and their aflatoxins biosynthesis in vitro and in situ conditions.

The purpose of this study was to investigate the effects of certain essential oils (star anise, lemon leaves, marjoram, fennel, and lavender) on the fungal growth of Aspergillus flavus and Aspergillus parasiticus and their production of aflatoxin B1 (AFB1). The degree of suppression of the aflatoxigenic strains' growth and their production of AFB1 is mainly affected by the kind and the concentration of the tested essential oils (EOs). Star anise essential oil had the lowest minimum inhibitory concentration (0.5 and 1.0 μL/mL) against A. flavus and A. parasiticus, respectively, so it was the best among the five different oils. The study of liquid chromatography with tandem mass spectrometry revealed that star anise EO resulted in a 98% reduction in AFB1 without a breakdown of AFB1 products after treatment thus the complete removal of AFB1 was done without any toxic residues. The combination showed a synergistic effect, the combinational treatment between γ-irradiation at a low dose (2 kGy) and star anise EO at concentrate 0.5 μL/g destroyed A. flavus and A. parasiticus inoculated (individually) in sorghum and peanut, respectively throughout the storage period (8 weeks).

Assessing the Levisticum officinale Koch. essential oil as a novel preservative for stored chia seeds (Salvia hispanica L.) with emphasis on probable mechanism of action.

The present study was undertaken to explore the inhibitory effect of Levisticum officinale Koch. essential oil (LoffEO) on the growth and aflatoxin B1 secretion by Aspergillus flavus (AF-LHP-SH1, aflatoxigenic strain) causing deterioration of stored chia seeds (Salvia hispanica). The chemical profile analysis of LoffEO by GC-MS analysis revealed the presence of α-terpinyl acetate (26.03 %) as a major component followed by terpineol <1- > (24.03 %) and citronellal (24.03 %). Results on antifungal and antiaflatoxigenic activity indicated that LoffEO at 2.0 and 1.75 μL/mL caused complete inhibition of growth and aflatoxin B1 production, respectively. Antifungal toxicity of LoffEO was strongly correlated with the inhibition of ergosterol content, leakage of cellular ions, and disintegration of membrane permeability. Reduction in cellular methylglyoxal by LoffEO indicated a novel antiaflatoxigenic mechanism of action. The LoffEO showed moderate free radical quenching activity in DPPH assay (IC50 = 26.10 μL/mL) and exhibited remarkable inhibitory efficacy against lipid peroxidation of chia seeds. In addition, LoffEO presented strong in situ antiaflatoxigenic efficacy, and exhibited non-phytotoxic nature, acceptable sensory characteristics, and favorable safety profile (LD50 = 19786.59 μL/kg), which recommends its practical utilization as a novel and safe preservative to improve the shelf life of stored chia seeds from fungal infestation and aflatoxin B1 contamination.

Bio-competitive exclusion: efficacy of non-aflatoxigenic Aspergillus section Flavi-L morphotypes in control of aflatoxigenic Aspergillus flavus in groundnuts (Arachis hypogaea L.)

BackgroundThe biological control mechanism of Aspergillus flavus (aflatoxigenic) strains in groundnuts with atoxigenic strains from the same species through competitive exclusion employed the use of endemic and well-adapted strains within the agro-ecological zones of Zimbabwe. The selected elite non-aflatoxigenic isolates of A. flavus native to Zimbabwe were evaluated for their capability to reduce aflatoxin contamination in groundnuts under laboratory conditions.ResultsAverage reduction percentages in aflatoxin B concentration for the 2019 and 2020 set of experiments ranged from 91.6 ± 3.4 to 95.8 ± 3.1% and 90.29 ± 3.6% to 95.29 ± 4.1%, respectively. Levels of aflatoxin in the co-inoculation research experiments administered were significantly reduced in all the experimental units carried out. Treatment efficiencies of the tested isolates in this study at 4:1 and 2:1 ranged from 1.20 to 2.52 and from 1.02 to 1.21, respectively. The efficacy of the tested non-aflatoxigenic strains against the aflatoxigenic strain native to Zimbabwe (ZMW 0127) indicates that the non-aflatoxigenic isolates of A. flavus. have sound practical applications against vast communities of aflatoxin-producing fungi across all the agro-ecological zones in Zimbabwe.ConclusionThe recognized non-aflatoxigenic isolates will be of an incentive as dynamic active ingredients in biocontrol formulations for the decrease in aflatoxins in groundnuts grown in Zimbabwe.

Chromatographic Analysis of Aflatoxigenic Aspergillus flavus Isolated from Malaysian Sweet Corn

High-performance liquid chromatography (HPLC) provides a quick and efficient tool for accurately characterizing aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. This method also provides a quantitative analysis of AFs in Aspergillus flavus. The method’s recovery was assessed by spiking a mixture of AF at different concentrations to the testing medium. The validity of the method was confirmed using aflatoxigenic and non-aflatoxigenic strains of A. flavus. The HPLC system, coupled with a fluorescence detector and post-column photochemical reactor, showed high sensitivity in detecting spiked AFs or AFs produced by A. flavus isolates. Recovery from medium spiked with 10, 20, 60, and 80 ppb of AFs was found to be 73–86% using this approach. For AFB1 and AFB2, the limit of detection was 0.072 and 0.062 ppb, while the limit of quantification was 0.220 and 0.189 ppb, respectively. The AFB1 concentrations ranged from 0.09 to 50.68 ppb, while the AFB2 concentrations ranged between 0.33 and 9.23 ppb. The findings showed that six isolates produced more AFB1 and AFB2 than the acceptable limit of 5 ppb. The incidence of aflatoxigenic isolates of A. flavus in sweet corn and higher concentrations of AFB1 and AFB2 emphasize the need for field trials to explore their real potential for AF production in corn.

The potential role of fungal volatile organic compounds in Aspergillus flavus biocontrol efficacy

Pre-harvest application of inherently non-aflatoxigenic Aspergillus flavus strains is an effective biocontrol strategy to minimize aflatoxin contamination of agricultural commodities, but researchers still do not fully understand how A. flavus biocontrol strains establish control over aflatoxigenic fungi. In this in vitro study, we tested the potential for A. flavus volatile organic compounds (VOCs), identified from a previous study, to serve as regulators of mycotoxin production. We exposed four Aspergillus strains (one non-aflatoxigenic and three aflatoxigenic) to five VOCs unique to aflatoxigenic A. flavus, and five VOCs unique to non-aflatoxigenic A. flavus, to study their impacts on growth as well as production of two mycotoxins: aflatoxin and cyclopiazonic acid (CPA). We found that growth of the fungi was not severely impacted by VOCs. However, toxin production was greatly affected and highly variable between strains and VOCs. Two non-aflatoxigenic VOCs (3-octanone and trans-2-methyl-2-butenal) significantly reduced aflatoxins in all aflatoxigenic strains. Two other non-aflatoxigenic VOCs (2,3-dihydrofuran and decane) significantly reduced aflatoxin levels in all aflatoxigenic strains and each VOC completely inhibited CPA production in one aflatoxigenic strain. Our findings offer evidence that the mechanism of control implemented by biocontrol strains may involve chemosensing through the production of one or more VOCs. These findings can be used to identify natural selectable markers for improved biocontrol strains or to supplement current biocontrol efforts through post-harvest exposure to VOC-related chemicals that mimic the presence of biocontrol strains.

PCR-Based Detection of Aflatoxogenic Strains of Aspergillus flavus Isolated from Poultry Feed

Twenty five isolates of A. flavus were detected by UV light (365nm) and ammonia vapor procedures. Aflatoxigenic A. flavus on Coconut Agar Medium (CAM) colored the reverse of glass petri dish with blue –green under UV light and produce a pink to red color by exposure to ammonia vapor. The detection by fluorescent blue revealed that 13 (52%) of isolates were aflatoxigenic by produce fluorescent color under UV (365nm) light, and also 13 (52%) of isolates were aflatoxigenic (positive) by ammonia vapor test. The molecular assessment was done on 25 isolates of A. flavus by using aflR gene primers. Five isolates of aflatoxigenic A. flavus positive identified isolates by PCR were randomly selected to sequence and analyze by basic local alignment search tool analysis (BLAST) to confirm the aflatoxigenic strains. Five isolates were positive and confirmed approximately compatible (99%-100%) homology with other A. flavus strains on NCBI.

Co-Inoculation of Aflatoxigenic and Non-Aflatoxigenic Strains of Aspergillus flavus to Assess the Efficacy of Non-Aflatoxigenic Strains in Growth Inhibition and Aflatoxin B1 Reduction

The pre-harvest biocontrol approach currently used includes laboratory inoculations using non-aflatoxigenic strains of Aspergillus flavus. This strategy effectively suppresses the indigenous aflatoxigenic strains and reduces aflatoxin accumulation in sweetcorn. The current in vitro study’s main objective is to determine the diametric growth rates of both Aflatoxin (AF)+ and AF− strains and improve the understanding of competitive relationships among these strains in sweetcorn (Zea mays). Sweetcorn kernels inoculated with AF+ strains only, AF− strains only, and co-inoculated with AF+ + AF− strains were investigated for aflatoxin concentrations. The diametric growth results revealed that growth rates of AF− strains at 25 and 30 °C were much greater than AF+ strains, which was in line with previous studies. The in vitro findings showed that the AKR5− and AKL34− biocontrol strains effectively inhibited the colony propagation and subsequent AFB1 contamination (up to 79%) of AF+ strains. On the other hand, the AKR1− and AKL35− were least effective in reducing AFB1 contents only by 58% and 60%, respectively. There was a significant difference (p &lt; 0.05) in the reduction of AFB1 contents achieved by AF− strains of A. flavus. The findings of the present study indicated the reduction in AFB1 with population expressions of AF+ strains by the AF− strains and supports the notion of competitive exclusion through vigorous development and propagation of the non-aflatoxigenic fungi.

Development of PCR, LAMP and qPCR Assays for the Detection of Aflatoxigenic Strains of Aspergillusflavus and A. parasiticus in Hazelnut.

Aspergillus flavus and A. parasiticus are two species able to produce aflatoxins in foodstuffs, and in particular in hazelnuts, at harvest and during postharvest phase. As not all the strains of these species are aflatoxin producers, it is necessary to develop techniques that can detect aflatoxigenic from not aflatoxigenic strains. Two assays, a LAMP (loop-mediated isothermal amplification) and a real time PCR with TaqMan® probe were designed and validated in terms of specificity, sensitivity, reproducibility, and repeatability. The capability of the strains to produce aflatoxins was measured in vitro and both assays showed to be specific for the aflatoxigenic strains of A. flavus and A. parasiticus. The limit of detection of the LAMP assay was 100–999 picograms of DNA, while the qPCR detected 160 femtograms of DNA in hazelnuts. Both techniques were validated using artificially inoculated hazelnuts and naturally infected hazelnuts. The qPCR was able to detect as few as eight cells of aflatoxigenic Aspergillus in naturally infected hazelnut. The combination of the LAMP assay, which can be performed in less than an hour, as screening method, with the high sensitivity of the qPCR, as confirmation assay, is able to detect aflatoxigenic strains already in field, helping to preserve the food safety of hazelnuts.

Natural Occurrence of Aflatoxigenic &lt;i&gt;Aspergillus&lt;/i&gt; Species and Aflatoxins in Traditional Korean Fermentation Starters, &lt;i&gt;Meju&lt;/i&gt; and &lt;i&gt;Nuruk&lt;/i&gt;

Meju and nuruk (respectively soybean and malt) are traditional Korean fermentation starters that are vulnerable to contamination by harmful microorganisms such as aflatoxigenic fungi and their associated aflatoxins (AFs). In this study, Aspergillus spp. were isolated and identified from a total of 57 meju and 18 nuruk samples collected from Korean markets. Their potential aflatoxigenicity was investigated by examining the presence of three aflatoxin biosynthetic genes (aflO, aflP, and aflR) using multiplex polymerase chain reaction (mPCR) assays. Thereafter, aflatoxin production of isolates and the natural occurrence of AFs in meju and nuruk samples were analyzed by high-performance liquid chromatography (HPLC). A total of 177 Aspergillus isolates were identified and 130 isolates were obtained from meju samples. Of these, 25 isolates (19.2%) contained all three aflatoxin biosynthetic genes, and five (20%) of these isolates produced aflatoxins. Forty-seven of the Aspergillus isolates were obtained from nuruk samples, five of which (10.6%) expressed all three AF biosynthetic genes; however, none of these strains produced AFs. HPLC analysis showed that 88% (51/58) of the meju samples and 39% (7/18) of nuruk samples were not contaminated with AFs (below limit of detection). Among the isolates isolated from meju and nuruk, there were aflatoxigenic strains containing all three aflatoxin biosynthetic genes or producing aflatoxin in medium, but the frequency of aflatoxin contamination was low in the meju and nuruk samples.

Efficacy of Cinnamomum cassia essential oil against food-borne molds and aflatoxin B1 contamination

The present study deals with the antifungal and antiaflatoxigenic potential of Cinnamomum cassia essential oil (CCEO) against aflatoxigenic food borne Aspergillus flavus strain (AF-LHP-PE-4), and probable mode of action including the safety profile on male mice. CCEO completely inhibited fungal growth and aflatoxin B1 secretion at its minimum inhibitory concentration (0.06 µL/mL). CCEO inhibited ergosterol biosynthesis in cell membranes and also disturbed the membrane fluidity. Significant enhancement in leakage of cellular ions and 260 and 280 nm absorbing cellular materials in response to increased concentrations of CCEO indicated the plasma membrane as the probable site of antifungal toxicity. The antiaflatoxigenic potency of CCEO was confirmed in terms of reduction in the level of cellular methylglyoxal (MG), the inducer of aflatoxin. Antioxidant activity of CCEO was confirmed through DPPH free radical scavenging activity as well as total phenolic content. Chemical profiling of CCEO by gas chromatography-mass spectrometry (GC-MS) analysis revealed the presence of cinnamaldehyde (84.01%) as the most abundant compound. CCEO exhibited a high LD50 (10410.75 µL/kg) on male mice, strengthening its favorable safety profile. This is the first report on CCEO as novel green preservative against food storage molds and aflatoxin B1 secretion.

Copper-Chitosan Nanocomposite Hydrogels Against Aflatoxigenic Aspergillus flavus from Dairy Cattle Feed.

The integration of copper nanoparticles as antifungal agents in polymeric matrices to produce copper polymer nanocomposites has shown excellent results in preventing the growth of a wide variety of toxigenic fungi. Copper-chitosan nanocomposite-based chitosan hydrogels (Cu-Chit/NCs hydrogel) were prepared using a metal vapor synthesis (MVS) and the resulting samples were described by transmission electron microscopy (TEM), X-ray fluorescence analysis (XRF), and small-angle X-ray scattering (SAXS). Aflatoxin-producing medium and VICAM aflatoxins tests were applied to evaluate their ability to produce aflatoxins through various strains of Aspergillus flavus associated with peanut meal and cotton seeds. Aflatoxin production capacity in four fungal media outlets revealed that 13 tested isolates were capable of producing both aflatoxin B1 and B2. Only 2 A. flavus isolates (Af11 and Af 20) fluoresced under UV light in the A. flavus and parasiticus Agar (AFPA) medium. PCR was completed using two specific primers targeting aflP and aflA genes involved in the synthetic track of aflatoxin. Nevertheless, the existence of aflP and aflA genes indicated some correlation with the development of aflatoxin. A unique DNA fragment of the expected 236 bp and 412 bp bands for aflP and aflA genes in A. flavus isolates, although non-PCR fragments have been observed in many other Aspergillus species. This study shows the antifungal activity of Cu-Chit/NCs hydrogels against aflatoxigenic strains of A. flavus. Our results reveal that the antifungal activity of nanocomposites in vitro can be effective depending on the type of fungal strain and nanocomposite concentration. SDS-PAGE and native proteins explain the apparent response of cellular proteins in the presence of Cu-Chit/NCs hydrogels. A. flavus treated with a high concentration of Cu-Chit/NCs hydrogels that can decrease or produce certain types of proteins. Cu-Chit/NCs hydrogel decreases the effect of G6DP isozyme while not affecting the activity of peroxidase isozymes in tested isolates. Additionally, microscopic measurements of scanning electron microscopy (SEM) showed damage to the fungal cell membranes. Cu-Chit/NCS hydrogel is an innovative nano-biopesticide produced by MVS is employed in food and feed to induce plant defense against toxigenic fungi.

Occurrence of aflatoxin- and ochratoxin A-producing Aspergillus species in Thai dried chilli

The present study aimed to examine the presence of aflatoxin- and ochratoxin A (OTA)-producing fungi in dried chilli ( Capsicum spp.). A total of 120 chilli samples, including 60 samples each of chilli powder and dried chilli pods, were examined for fungal contamination. Results showed that 86.7% and 96.7% of chilli powder and dried chilli pods, respectively, were contaminated by fungi at a concentration of <10 3 CFU/g. Overall, 500 fungal isolates were recovered and identified using a morphological method. The most frequent strain was Aspergillus section Flavi (46.6%), followed by Aspergillus section Nigri (34%), Penicillium (7.6%), Aspergillus section Aspergillus (7.6%), Aspergillus section Circumdati (2.2%) and Rhizopus (2%). In total, 37.5% and 43.8% of Aspergillus section Flavi isolates obtained from dried chilli powder and dried chilli pods, respectively, produced aflatoxin. Of the 96 aflatoxigenic isolates, 96.9% of the strains produced aflatoxin B and 3.1% produced both aflatoxin B and G. However, β-tubulin sequence analysis revealed that all aflatoxin-producing isolates were identical to A. flavus . Moreover, 170 isolates of Aspergillus section Nigri , 11 of Aspergillus section Circumdati , 2 of Aspergillus section Flavi producing yellow-to-cinnamon colonies and 38 of Penicillium spp. were examined for their ability to produce OTA. In total, 1 isolate of Aspergillus section Nigri and 2 isolates of Aspergillus section Flavi produced OTA. The first isolate was identified as A. carbonarius , whereas the latter two isolates were both A. alliaceus . The highest OTA production by A. carbonarius (622.4 ng/g) was observed following incubation on MEA at 25 °C for 21 days. Conversely, the two A. alliaceus isolates produced OTA at levels of 583.7 and 743.7 ng/g, respectively, after cultivation on YES at 25 °C for 21 days. To the best of our knowledge, this is the first report regarding the natural occurrence of aflatoxin- and OTA-producing fungi in dried chilli from Thailand. • More than 95% of dried chilli powder and dried chilli pods collected in Thailand was contaminated with fungi. • Aspergillus section Flavi is the most common species in dried chilli. • Aspergillus section Flavi in dried chili samples is frequently aflatoxigenic strain. • Ochratoxin A producing species, A. carbonarius and A. alliaceus , were detected in dried chilli powder.

Mechanism of inhibition of aflatoxin synthesis by non-aflatoxigenic strains of Aspergillus flavus

Aflatoxins are toxic secondary metabolites primarily produced by Aspergillus flavus and A. paraciticus. Exposure to these mycotoxins through contaminated food and feed may cause oxidative stress and liver toxicity in animals. One of the promising strategies to mitigate aflatoxin accumulation is the biological management during pre-harvest using non-aflatoxigenic A. flavus. The mechanism offered by these strains in mitigating aflatoxin is still unclear. Thus, the aim of the present study is to delineate the mechanism of intraspecific inhibition of aflatoxin production. Among the 18 non-aflatoxigenic strains evaluated, six strains were able to reduce more than 50% of the aflatoxins produced by the native aflatoxigenic strains. The non-aflatoxigenic strains used in this study failed to degrade the aflatoxins. Eventhough, the non-aflatoxigenic strains were not able to inhibit the synthesis of aflatoxins completely. Four non-aflatoxigenic isolates could competitively excluded the aflatoxigenic strain. Furthermore, when non-aflatoxigenic and an aflatoxigenic isolate were separated by 0.4 and 3 μm filters, aflatoxin synthesis was not significantly reduced. However, when the pore size was 8 μm, there was a significant decrease in aflatoxin production. This results suggest the role of physical contact between the hyphae, thigmoregulation, in the inhibition of aflatoxin production. Additionally, to better understand the transcriptional level control of this phenomenon, we analyzed the gene expression profile of aflatoxin biosynthesis genes in the aflatoxigenic strain. The aflatoxin biosynthesis genes were down regulated in the aflatoxigenic strain in contact with non-aflatoxigenic strain group when compared to the control. This is the first evidence of the combined action of competitive exclusion and thigmodownregulation which led to the intraspecific inhibition of aflatoxin production.

Myristica fragrans essential oil nanoemulsion as novel green preservative against fungal and aflatoxin contamination of food commodities with emphasis on biochemical mode of action and molecular docking of major components

The study reports the antifungal and antiaflatoxigenic mechanism of action of chitosan fabricated Myristica fragrans essential oil nanoemulsion (Nm-MFEO) against an aflatoxigenic strain of Aspergillus flavus (AF LHP R14). Significant inhibition in ergosterol biosynthesis by Nm-MFEO indicated plasma membrane as potential target site of antifungal action. Moreover, Nm-MFEO caused dose dependent inhibition of methylglyoxal (aflatoxin inducer) biosynthesis suggesting novel antiaflatoxigenic mechanism of action. Significant impairment in enzymatic (superoxide dismutase and catalase) and non-enzymatic (oxidized and reduced glutathione and ratio of oxidized and reduced glutathione) anti-oxidative defense molecules in AF LHP R14 cells after treatment with Nm-MFEO suggested biochemical mechanism of action. Nm-MFEO exhibited promising antioxidant activity as determined through DPPH˙ and ABTS˙+ assay with IC50 value 1.20, 0.157 μL/mL, respectively and higher phenolic content (11.87 μg/mg gallic acid). Further, the in silico molecular docking of major components of MFEO viz. myristicin, elemicin and thujanol with aflatoxin producing regulatory genes Ver 1 and Omt A depicted important molecular site of interaction supporting the applicability of MFEO nanoemulsion as plant based green food preservative. The present study is the first report on the effect of chitosan fabricated MFEO nanoemulsion resulting in the inhibition of methylglyoxal, thereby suggesting its future exploitation for the development of aflatoxin resistant food crop varieties through green transgenics.

Rapid isolation of non-aflatoxigenicAspergillus flavus strains

In the present study, a method for screening non-aflatoxigenicAspergillus flavus in soil samples collected from major peanut-growing regions of China was developed. The single colonies were picked and cultured onAspergillus flavus andparasiticus agar (AFPA). If the reverse side of the colony on AFPA was orange-coloured, it was consideredA. flavus orAspergillus parasiticus. After the genomic DNA of each strain was extracted, 28S rRNA and calmodulin were amplified and sequenced to determine the species. The key gene,aflR, was amplified and digested via polymerase chain reaction-restriction fragment length polymorphism. The aflatoxigenicA. flavus and the non-aflatoxigenicA. flavus andA. parasiticus were distinguished by enzyme digestion ofaflR. 156 strains ofA. flavus were screened, which consisted of 135 aflatoxigenic and 21 non-aflatoxigenic strains. The aflatoxin producing ability of each strain was confirmed using solid-state fermentation experiments. Using the method developed in the present study, we confirmed that the non-aflatoxigenicA. flavus strains isolated lost their capacity to produce aflatoxins. Considering there could be some alterations in other functional genes, some non-aflatoxigenic strains could be identified inaccurately as aflatoxigenic strains, although that did not occur in the present study. The growth of non-aflatoxigenicA. flavus was observed, and the most rapidly growing non-aflatoxigenic strain was selected for plate confrontation assays and toxic mixed culture experiments. The inhibition rate of non-aflatoxigenicA. flavus against aflatoxigenicA. flavus was 55.4 and 72.6% in potato dextrose agar (PDA) plate and natural soybean medium, respectively. The screened non-aflatoxigenicA. flavus strains provide a microbial resource for biological control of aflatoxin contamination.

Efficacy of Streptomyces philanthi RL-1-178 culture filtrate against growth and aflatoxin B1 production by two aflatoxigenic fungi on maize seeds

The main objective of this study was to investigate the antifungal activities of a culture filtrate from Streptomyces philanthi RL-1-178 (culture filtrate RL-1-178) on growth and aflatoxin B1 production of Aspergillus parasiticus TISTR 3276 and A. flavus PSRDC-4 inoculated onto maize seeds. The culture filtrate RL-1-178 was first analyzed for its composition and found to contain 86 compounds with acetic acid (53.59%) as the major component. It could inhibit both the growth and the production of aflatoxin B1 (AFB1) of the two aflatoxigenic species. Complete prevention of AFB1 production was achieved with 5% (v/v) of the culture filtrate RL-1-178. It also inhibited spore germination depending on the duration of treatment. The culture filtrate RL-1-178 altered on the cell wall composition of the two aflatoxigenic fungal strains by causing loss of cytoplasm and distortion of the plasma membranes evident by transmission electron microscopy (TEM) image. For testing on maize seeds, the culture filtrate RL-1-178 at 30% (v/w of maize seeds) exhibited 100% and 98% inhibition on AFB1 production by A. parasiticus TISTR 3276 and A. flavus PSRDC-4, respectively, without affecting maize seed emergence. The culture filtrate RL-1-178 possessed antifungal activity against the two aflatoxigenic fungal strains grown on maize seeds. The effective dose was 30% (v/w) with no effect on the maize emergence (100%). Our results suggest that the culture filtrate from S. philanthi RL-1-178 can be used as a natural product that controls the two aflatoxigenic fungi.

Influence of aflatoxin in Nuruk on the safety of starch-based alcoholic beverage.

Nuruk is a fermentation agent used to manufacture alcoholic beverages, which contains a variety of microorganisms. Most microorganisms in Nuruk are useful for the production of alcoholic beverages; however, Nuruk can be infected with Aspergillus flavus, which produces aflatoxin (AF). Therefore, this study analyzed total AF concentrations in Nuruk, the transfer of AF from Nuruk to alcoholic beverages, and AF-producing microorganisms to determine the safety of alcoholic beverages with respect to this toxin. ELISA showed that total AF levels in 14 of 61 Nuruk samples exceeded 15 ppb, the Korean permissible level in cereal products. In alcoholic beverages produced with Nuruk containing AF levels >15 ppb, only AF G1 was detected, at a level of 0.3 ppb, and the transfer ratio of AF G1 was approximately 1.2% to 1.3%. The dominant genera in Nuruk were Aspergillus and Rhizopus. Among 30 strains belonging to Aspergillus, 10 produced only AF B1 at levels of 0.1 to 2.4 ppb after incubation at 25°C for 8 days on potato dextrose agar plates. Although AF in Nuruk was rarely transferred to alcoholic beverages and the aflatoxigenic strains were found to possess poor AF-producing capacity, further efforts to reduce AF in Nuruk are needed to ensure the safety of alcoholic beverages.

Application and Evaluation of the Loop-Mediated Isothermal Amplification assay for the Detection of Aflatoxigenic Fungi Contaminants of Rice Grains in Kenya

Aflatoxigenic fungi are most common filamentous fungi that synthesis aflatoxins and represent the major fungal pathogens to agricultural products. Aflatoxins remain a major threat to global food security, these molecules could be resisted into food during processing and in additional may remain within the food chain. Aflatoxins are carcinogenic, hepatotoxic, mutagenic, teratogenic, can inhibit numerous metabolic systems and immunosuppressive properties. Studies of aflatoxigenic strains can help to enhance strategies control and prevent aflatoxigenic fungi contamination and aflatoxins production in foodstuffs. In this study, isolation of Aspergillus species was based on morphological characteristics including the mycelium growth pattern, color, and properties of fruiting bodies of the fungi. The innovated technique loop-mediated isothermal amplification assay was applied to amplify Norsolorinic Acid gene. The loop-mediated isothermal amplification have been optimized by combination of the rapidity, simplicity and specificity to detect the target genomic DNA in the reactions. The amplification curves monitored by Loopamp realtime Turbidimeter were analyzed in order to distinguish aflatoxigenic and non-aflatoxigenic strains. Overall, the results showed that the loop-mediated isothermal amplification method was effective in detecting aflatoxigenic strains with high specificity of 71.5% and sensitivity under lower concentrations of DNA. In additional, it was faster than the conventional polymerase chain reaction. The loop-mediated isothermal amplification assay described in this study might be a promising tool for prediction potential threats by aflatoxigenic fungi and aflatoxins risk in food and commodities.

Toxigenicity and phylogeny of Aspergillus section Flavi in poultry feed in Iran.

Background and Purpose:This study was conducted to evaluate the presence of aflatoxigenic strains and level of aflatoxin in poultry feed. Aflatoxigenic strains were investigated in corn and soybean meal as the ingredients of poultry feed, as well as in two types of commercial feed, namely pellet and mash. The gene sequencing was performed to identify the species of Aspergillus section Flavi.Materials and Methods:All samples were randomly collected from feed storage silos located in Iran in 2018. The samples were cultured on specialized media for 2 weeks at 28ºC. Identification of Aspergillus section Flavi isolates was based on macro- and microscopic morphological criteria and molecular analysis. The thin-layer chromatography (TLC) was applied to confirm the aflatoxigenic isolates. In addition, the level of aflatoxin B1 (AFB1) produced by these isolates was determined by high-performance liquid chromatography. The strains were subjected to sequence analysis, and Bt2 PCR products were purified by the QIAquick PCR purification kit. At the final stage, the phylogenetic tree was built.Results:Among 54 isolates identified as Aspergillus section Flavi, 20 (37%) isolates were found to produce aflatoxin at a range of 11.28±1.18 to 2239.92±92.26 µg/g fungal dry weight. The aflatoxigenic isolates had the frequencies of 45%, 40%, 10%, and 5% in the corn, pellet, soybean meal, and mash samples, respectively. Furthermore, the mean concentrations of AFB1 were significantly higher in the corn samples (707.04±39.05) than that of other poultry feed samples (P<0.05). A total of 34 (63%) isolates were detected as non-aflatoxigenic on the yeast extract-sucrose broth in TLC analysis. The toxigenic isolates produced the highest (2232.62±55.49) and lowest (11.28±1.18) levels of AFB1 in the corn samples, compared to other feedstuffs. Furthermore, the mean level of AFB1 in mash product was 554.09±10.36 µg/g, compared to a mean level of 229.22±11.09 µg/g in pellets. The isolates were randomly selected, sequenced, and then analyzed. Subsequently, the phylogenetic tree of Aspergillus section Flavi was plotted.Conclusion:The process of converting raw ingredients to compound poultry feed is more hazardous when there is not enough time and temperature provided to eliminate aflatoxigenic isolates. Therefore, Aspergillus section Flavi in poultry feed can pose a threat to the poultry industry and poultry products, thereby affecting the health status of humans. Unprocessed/processed materials, such as corns and pelleted feed, need further monitoring, especially when conditions are not optimal for destroying the fungus.