Aflatoxin Production Research Articles

Molecular identification and management of mycotoxigenic fungi in stored corn Grains

Mycotoxin-producing molds which considered as common maize grains contaminants are the genera Fusarium, Aspergillus and Penicillium. There are natural and safe ways to protect grains from mold contamination as the use of essential oils and chemical treatments. A total number of 25 samples were used to study the natural frequency in five governorates in Egypt, Molecular identification indicated that the most frequent fungi were Fusarium verticillioides, Aspergillus niger, Talaromyces verruculosus, Aspergillus flavus and Aspergillus terreus. The in vitro studies have been done to determine mycelial growth and spore germination inhibition of the two A. flavus; isolated and reference isolates. Thyme and acetic acid were tested in direct contact assay to study their effects on mycelial growth. Treatments showed significant impact on mycelial growth and spore germination inhibition of both A. flavus isolates. In the postharvest application treatments: as vapour and carrier contact assay, Thyme and Acetic acid were tested to determine their influence on growth and aflatoxin production in A. flavus isolates by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC–ESI–MS/MS). Results indicated that both treatments were effective in inhibition of aflatoxin production in both vapour and carrier assays as they succeeded in reducing AFB1 while they inhibited completely the production of AFB2. The extent of the inhibition of aflatoxin production was dependent on the concentration and storage duration of treatments applied.

Inhibition of Aflatoxin Production in Aspergillus flavus by a Klebsiella sp. and Its Metabolite Cyclo(l-Ala-Gly).

During an experiment where we were cultivating aflatoxigenic Aspergillus flavus on peanuts, we accidentally discovered that a bacterium adhering to the peanut strongly inhibited aflatoxin (AF) production by A. flavus. The bacterium, isolated and identified as Klebsiella aerogenes, was found to produce an AF production inhibitor. Cyclo(l-Ala-Gly), isolated from the bacterial culture supernatant, was the main active component. The aflatoxin production-inhibitory activity of cyclo(l-Ala-Gly) has not been reported. Cyclo(l-Ala-Gly) inhibited AF production in A. flavus without affecting its fungal growth in a liquid medium with stronger potency than cyclo(l-Ala-l-Pro). Cyclo(l-Ala-Gly) has the strongest AF production-inhibitory activity among known AF production-inhibitory diketopiperazines. Related compounds in which the methyl moiety in cyclo(l-Ala-Gly) is replaced by ethyl, propyl, or isopropyl have shown much stronger activity than cyclo(l-Ala-Gly). Cyclo(l-Ala-Gly) did not inhibit recombinant glutathione-S-transferase (GST) in A. flavus, unlike (l-Ala-l-Pro), which showed that the inhibition of GST was not responsible for the AF production-inhibition of cyclo(l-Ala-Gly). When A. flavus was cultured on peanuts dipped for a short period of time in a dilution series bacterial culture broth, AF production in the peanuts was strongly inhibited, even at a 1 × 104-fold dilution. This strong inhibitory activity suggests that the bacterium is a candidate for an effective biocontrol agent for AF control.

Macro-micro exploration on dynamic interaction between aflatoxigenic Aspergillus flavus and maize kernels using Vis/NIR hyperspectral imaging and SEM technology

Aspergillus flavus and its toxic metabolites-aflatoxins infect and contaminate maize kernels, posing a threat to grain safety and human health. Due to the complexity of microbial growth and metabolic processes, dynamic mechanisms among fungal growth, nutrient depletion of maize kernels and aflatoxin production is still unclear. In this study, visible/near infrared (Vis/NIR) hyperspectral imaging (HSI) combined with the scanning electron microscope (SEM) was used to elucidate the critical organismal interaction at kernel (macro-) and microscopic levels. As kernel damage is the main entrance for fungal invasion, maize kernels with gradually aggravated damages from intact to pierced to halved kernels with A. flavus were cultured for 0–120 h. The spectral fingerprints of the A. flavus-maize kernel complex over time were analyzed with principal components analysis (PCA) of hyperspectral images, where the pseudo-color score maps and the loading plots of the first three PCs were used to investigate the dynamic process of fungal infection and to capture the subtle changes in the complex with different hardness of the maize matrix. The dynamic growth process of A. flavus and the interactions of fungus-maize complexes were explained on a microscopic level using SEM. Specifically, fungus morphology, e.g., hyphae, conidia, and conidiophore (stipe) was accurately captured on the microscopic level, and the interaction process between A. flavus and nutrient loss from the maize kernel tissues (i.e., embryo, and endosperm) was described. Furthermore, the growth stage discrimination models based on PLSDA with the results of CCRC = 100 %, CCRV = 97 %, CCRIV = 93 %, and the prediction models of AFB1 based on PLSR with satisfactory performance (R2C = 0.96, R2V = 0.95, R2IV = 0.93 and RPD = 3.58) were both achieved. In conclusion, the results from both macro-level (Vis/NIR-HSI) and micro-level (SEM) assessments revealed the dynamic organismal interactions in A. flavus-maize kernel complex, and the detailed data could be used for modeling, and quantitative prediction of aflatoxin, which would establish a theoretical foundation for the early detection of fungal or toxin contaminated grains to ensure food security.

Potential of gaseous ozone to inhibit Aspergillus flavus and detoxify aflatoxin B1 in agar and broth media (solid and liquid food states) at different growth phases

Agricultural food crops are prone to Aspergillus flavus contamination during post-harvest and storage stages. Their consumption is toxic due to aflatoxin production, a potent carcinogenic and immunosuppressive agent. Present study explored the ozone's effect as a function of food media states (solid and liquid) to inhibit Aspergillus flavus under three modes of treatment (0th-day, Every-day and Nth-day ozone treatment), that adds novelty to the work. Results showed that ozone concentration to treat Aspergillus flavus and aflatoxins is higher in liquid than solid food state. Further, ozone treatment affected the intactness of fungal spores and inhibited 100% conidial growth. It also exhibited 88 %% inhibition and 90% reduced biomass in solid and liquid food states. Effectiveness of ozone towards inhibiting Aspergillus flavus and aflatoxin was in the following order: Nth -day treatment > Every-day treatment > 0th -day ozone treatment. Further, growth studies showed ozone treated cultures exhibit delayed sporulation and aflatoxin production by 72 h. when cultured on fresh media. Additionally, ozone purging into extracted aflatoxin led to > 99% degradation. The above results show the significance of ozone technology in increasing the shelf-life of post-harvested food crops. Further, it can be used as a disinfectant in the food industry to treat contaminated food crops/liquid products by detoxifying aflatoxins.

Acetyl-CoA carboxylase as potential molecular target of dimethyl trisulfide on inhibiting the Aspergillus flavus growth and aflatoxins production

Aspergillus flavus and the produced aflatoxins are common hazardous contaminants in grains and food, which generated great damage to food security and human health. Acetyl-CoA carboxylase (ACC), catalyzing acetyl-CoA to malonyl-CoA, is the first and speed-determining step in fatty acid biosynthesis (cell membrane composition) and aflatoxins biosynthesis process in A. flavus. Here, we demonstrated that dimethyl trisulfide (DMTS), emitted from bacteria, could interact with ACC, decrease the enzymatic activity from 377.62 to 191.42 kat/g in vitro, down-regulate the expression of ACC and other important genes in fatty acid and aflatoxin biosynthesis pathway, reduce the content of hexadecanoic acid, octadecanoic acid and eicosanoic acid, and finally prevent A. flavus growth and aflatoxins production. Two binding sites containing 19 amino acids were detected between DMTS and ACC through molecular docking analysis. These amino acids were highly conserved in different filamentous fungi, which accounted for the broad antifungal spectrum of DMTS. Therefore, we concluded that ACC is the molecular target of DMTS on A. flavus growth and aflatoxins production. These results elucidate the inhibitory mechanism of DMTS on fungal pathogens, and provide basic theories for the development of novel antifungal agents.

Determination of Mycotoxigenic Fungi and Total Aflatoxins in Stored Corn from Sites of Puebla and Tlaxcala, Mexico

This paper aimed to evaluate the contamination with mycotoxigenic fungi and total aflatoxins in stored corn from different sites in Puebla and Tlaxcala, Mexico. Methodology. The study was conducted at two sites in Puebla (San Salvador El Seco and Junta Auxiliar La Resurrección) and two sites in Tlaxcala (Tlaltepango and Nativitas). A total of 80 samples of stored corn were collected. Identification of Aspergillus flavus was performed by microculture techniques and specific taxonomic keys (macromorphological and micromorphological). Then, samples of contaminated corn were selected, and aflatoxin production was confirmed using a direct solid-phase ELISA kit. A total of 25 A. flavus strains were identified. Other possible mycotoxin-producing fungi were Penicillium (n=52) and Fusarium (n=19). Regarding total aflatoxin contamination, all samples were contaminated within a range of 1.589 to 11.854 μg/kg, and the average concentration was 6.3 μg/kg corn. Implications. The detection of mycotoxigenic fungi in the samples tested and of aflatoxins in corn highlights the importance of monitoring these fungi. Since food safety is at risk, it shows the need for methods to control these fungi and their metabolites.

Almond defect and freshness inspection system using hyperspectral imaging and deep learning techniques

Almonds, recognized as a nutritious and low-calorie snack rich in unsaturated fats and essential nutrients, are primarily imported from California. Despite undergoing grading control by the United States Department of Agriculture (USDA), concerns persist regarding the presence of defective kernels. Manual hand-picking is the conventional method for almond processing; however, prolonged picking can reduce accuracy due to visual fatigue. Consumers prioritize freshness due to fungi’s potential for aflatoxin production, emphasizing the need for vigilant almond consumption. In response to these challenges, this study presents an innovative approach that employs a lightweight VGG16 and an enhanced MobileNet model for identifying defects based on RGB images of almonds. This method effectively classifies broken, scratched, and malformed kernels. Additionally, hyperspectral imaging (HSI) data in the 400–1000 nm range were collected to distinguish insect-damaged from undamaged kernels and assess almond freshness. For comparative analysis, various band selection algorithms were applied to extract distinct spectral bands. For insect damage identification, we propose a merged CNN model created by simultaneously combining 1D- and 2D-CNNs, allowing for the concurrent extraction of spectral and spatial characteristics. This approach achieved a remarkable ACC of 0.9905 and a Kappa value of 0.9521. Meanwhile, in evaluating freshness, PCA was employed to select the first 30 principal components as vital bands. Our proposed lightweight 1D-CNN delivered an ACC of 0.9768 and a Kappa value of 0.9527 in freshness assessment.

Inhibitory Effects of Bacillus subtilis Isolated from Meju (Fermented Soybean Brick) on the Growth of Aspergillus parasiticus

Background: Meju is a base material for making soy sauce, soybean paste, and red chili pepper paste, which are representative ingredients of Korean cuisine. Objectives: This study aimed to isolate a predominant bacterial strain of B. subtilis from meju and to observe its inhibitory effects on an aflatoxigenic mold. Methods: We used yellow soybeans (Glycine max (L.) Merr.) grown in South Korea, and meju was produced according to the recommended methods of the Korea Food Research Institute. The identification of the strain was conducted based on its morphological and biochemical characteristics and 16S rDNA sequence. Evaluation of the bacterial effect against A. parasiticus ATCC 15517 was done in yeast extract–sucrose broth at 28 °C. Its inhibitory effect was evaluated using two approaches: mycelial weight and aflatoxin production. Aflatoxins were determined using high-performance liquid chromatography. Results: In the meju samples fermented for three months, a B. subtilis strain, K-0924 was identified. At the end of the incubation period of A. parasiticus, dry mycelial weight was significantly reduced by more than 80% (p < 0.01) and total aflatoxin production was inhibited by more than 63% (p < 0.05) in the presence of B. subtilis. Conclusions: These results indicate that B. subtilis K-0924 inhibits the growth and aflatoxin production of toxigenic Aspergillus, which can be contaminated with meju. We could expect more inhibition by other bacteria related to fermentation of meju, and further examination is necessary on species other than B. subtilis.

The impact of fullerenol nanoparticles on the growth of toxigenic Aspergillus flavus and aflatoxins production in vitro and in corn flour.

Antifungal and antimycotoxigenic activity of fullerenol nanoparticles (FNPs) were investigated on Aspergillus flavus growth isolated from a real food sample and aflatoxins (AFs) (AFB1 and AFB2 ) production. The final FNPs concentrations in in vitro and in commercial corn flour after the stationary incubation period of 7 and 14 days were in the range 0.16-80µg/mL and 0.16-80µg/g, respectively. Nanocharacterization of FNPs revealed an average size of 5-20nm and a zeta potential of -35mV. The highest degree of A. flavus mycelium growth inhibition (28%) after 7 days was observed for applied FNP concentration of 8.0µg/mL, while after 14 days FNP concentration of 0.32µg/mL led to the maximal inhibition of A. flavus mycelium growth (36%). Spearman's correlations analysis revealed a strong positive correlation between AFB1 and AFB2 concentrations in YES broth after 7 (R=0.994, p<0.05) and 14 days (R=0.976), as well as between AFs concentrations and A. flavus mycelium mass after 7 (R=0.786 for AFB1 and R=0.766 for AFB2 ) and 14 days (R=0.810 for AFB1 and R=0.833 for AFB2 ). Paired samples t-test showed the existence of a statistically significant difference (p<0.05) between the produced AFs concentrations after the incubation of 7 and 14 days. Regarding the artificially inoculated corn flour the lower applied FNP concentrations (0.16-0.8µg/g) achieved a reduction of AFB1 up to 42% and 60% after 7 and 14 days, respectively.

Aspergillus population diversity and its role in aflatoxin contamination of cashew nuts from coastal Kenya.

Cashew nuts are among the main cash crops in coastal Kenya, due in large part to their high nutritional value. Unfortunately, they also make them highly susceptible to mold contamination, resulting in biodeterioration of the nutritional value and potential contamination with toxic secondary metabolites, such as aflatoxins, that cause them to be rejected for sale at the market. We determined the population diversity of the Aspergillus species and their role in aflatoxin contamination in cashew nuts in selected coastal regions of Kenya. Fifty raw cashew nut samples were collected from post-harvest storage facilities across three counties in Kenya's coastal region and examined for moisture content and the presence of Aspergillus fungi. About 63 presumptive isolates were recovered from the cashew nuts. ITS and 28S rDNA regions were sequenced. The aflD, aflM and aflR genes were amplified to identify the potentially aflatoxigenic from the Aspergillus isolates. The Aflatoxins' presence on the isolates was screened using UV and the ammonia vapour test on coconut milk agar and validated using ELISA assay. A comparison of cashew moisture content between the three counties sampled revealed a significant difference. Sixty-three isolates were recovered and identified to section based on morphological characters and their respective ITS regions were used to obtain species identifications. Three sections from the genus were represented, Flavi and Nigri, and Terrei with isolates from the section Nigri having slightly greater abundance (n = 35). The aflD, aflM and aflR genes were amplified for all isolates to assess the presence of the aflatoxin biosynthesis pathway, indicating the potential for aflatoxin production. Less than half of the Aspergillus isolates (39.68%) contained the aflatoxin pathway genes, while 22.22% isolates were aflatoxigenic, which included only the section Flavi isolates. Section Flavi isolates identification was confirmed by calmodulin gene. The presence of species from Aspergillus section Flavi and section Nigri indicate the potential for aflatoxin or ochratoxin in the cashew nuts. The study established a foundation for future investigations of the fungi and mycotoxins contaminating cashew nuts in Kenya, which necessitates developing strategies to prevent infection by mycotoxigenic fungi, especially during the storage and processing phases.

Effects of High Concentration-Short Time and Low Concentration-Long Time Ozone Treatments on Growth and Aflatoxin B1 Production of Aspergillus flavus in Red Pepper

ABSTRACT The effects of high concentration-short time (HCST) and low concentration-long time (LCLT) gaseous ozone treatments on growth and aflatoxin B1 production of Aspergillus flavus in semi-dried red pepper are presented for the first time in this paper. Potato dextrose agar (PDA) media and peppers were inoculated with a toxigenic strain of A. flavus and exposed to either HCST (1500 μL L−1 for 1–8 h) or LCLT (0.1 μL L−1 for 8 d) ozone treatments. Both treatments significantly inhibited A. flavus growth in PDA and peppers (p < 0.05). In pepper samples, fungal count reductions obtained by HCST and LCLT ozone treatments were around 1.57 and 1.66 log, respectively. However, the inhibitory effect decreased in time which shows that the fungus rapidly develops a resistance to ozone. The amounts of aflatoxin B1 produced in PDA and pepper were also significantly reduced (by more than 85%) by the treatments (p < 0.05). These results demonstrate the potential of ozone treatments in preventing fungal contamination and aflatoxin production in red pepper.

Inhibition of Aflatoxin B1 by Aqueous extract from Green Cardamom

Aflatoxin B1 is a secondary metabolite produced by Aspergillus section Flavi during their development, is one of the most dangerous mycotoxins identified to date, that may contaminate many commodities, especially in tropical and sub-tropical regions. Aflatoxin B1 is believed to be the most essential member of this family of mycotoxins, due to its carcinogenic properties in humans and animals. However, there are many strategies to prevent mycotoxin contamination in which some of them are based on the use of natural compounds that may able to minimize the toxin synthesis pathways. Aims: The current work aimed to identify specific compounds from Green Cardamom that expected to interfere with aflatoxin production pathway. Methodology: The adapted method was based on the incubation of fungal culture with different concentrations of the aqueous extraction of Green Cardamom. Results: The aqueous extract of Green Cardamom was able to inhibit Fungal growth and Aflatoxin B1 production by the toxigenic strain of Aspergillus Flavus in a dose-dependent manner. Discussion: the reduction of Aflatoxin B1 production was significantly higher (94.34%) in comparison with other plant extracts such as Caffeine (50%), and reduced fungal growth with an average of (4%) compared to control. Conclusion: Aqueous Extracts of Green Cardamom show an obvious reduction in inhibition synthesis of Aflatoxin B1 by toxigenic Aspergillus strain, the adapted method was effective, but even further investigation is still recommended.

Early evaluation of aflatoxin occurrence risk using an ultra-sensitive immunoassay for pre-aflatoxin protein biomarker detection

The pre-aflatoxin protein biomarker PAB-01 is a key target for early predicting of aflatoxin occurrence risk. Improving its detection sensitivity is significant for the early evaluation of potential risks. In this study, we obtained the monoclonal antibody 6A6 against PAB-01 through hybridoma technology. A sandwich ELISA was developed using pairwise combinations of PAB-01 nanobody, monoclonal antibody, and polyclonal antibody. It was found that the nanobody as the capture antibody and monoclonal antibody as the detection antibody exhibited the highest sensitivity, with a detection limit of 0.92 ng/mL. Based on this, a rolling circle amplification-based immunoassay (RCA-EIA) was established, with a linear range of 0.056–41.152 ng/mL and a detection limit of 0.033 ng/mL. Recovery rates in spiked peanut and corn samples ranged from 94.49% to 127.50%. This method successfully identified the presence of the PAB-01 protein in maize samples 10 days prior to the aflatoxin production. Based on PAB-01 concentration classifications, the maize samples were categorized into different risk levels. This development is significant for early warning and risk stratification management of aflatoxin occurrence, providing a highly effective tool in ensuring food safety.

Characterization of Aspergillus section Flavi associated with stored grains

Increased frequencies of Aspergillus section Flavi and aflatoxins in cereal grains have been seen in recent years due to changes in climate circumstances, such as high temperatures and drought. To assess the microbiological risks of contamination, it is critical to have a reliable and accurate means of identifying the fungi. The main goal of this study was to characterize Aspergillus species from section Flavi obtained from twenty-three samples of barley and maize grains, gathered from different markets in Qena, Egypt, using morphological and molecular techniques. Twenty-three isolates were chosen, one isolate from each sample; they were identified as A. aflatoxiformans (4 isolates), A. flavus (18), and A. parasiticus (1). The existence of four aflatoxin biosynthesis genes was also investigated in relation to the strains’ ability to produce total aflatoxins and aflatoxin B1, focusing on the regulatory gene aflR and the structural genes aflD and aflM. All strains producing aflatoxins were linked to the presence of aflR1 and/or aflR2, except two isolates that exhibited aflatoxins but from which aflR1 or aflR2 were not detected, which may be due to one or more missing or unstudied additional genes involved in aflatoxin production. AflD and aflM genes were amplified by 10 and 9 isolates, respectively. Five samples of barley and maize were contaminated by aflatoxins. Fifteen isolates were positive for producing total aflatoxins in the range of 0.1–240 ppm. Antagonistic activity of Trichoderma viride against A. flavus (F5) was assessed at 31.3%. Trichoderma reduced total aflatoxins in all treated seeds, particularly those subjected to Trichoderma formulation.

Determination and Comparison of Total Aflatoxin Levels in Groundnuts from Four Major Markets in Lagos State, Nigeria

Aflatoxins (Aspergillus flavus toxins) are poisonous substances produced by some fungi on crops, foods, and feeds. The study was carried out to determine and to compare total aflatoxin levels in groundnuts sold in Lagos markets, Nigeria. Microbiological and toxicology tests were carried out on raw, boiled, and roasted groundnuts from the same source. The microbiological test was carried out to detect the fungi that produce aflatoxins as their secondary metabolite. The groundnuts samples were cultured on Potato Dextrose Agar (PDA). The isolates were identified using standard microbiological methods, characteristics such as presence of spores, shape, colour of spores and presence of septa were used in identification. Aspergillus flavus (75%; percentage of occurrence) was the most frequently isolated fungus from the groundnut samples. The other contaminant isolated from the samples were Aspergillus niger (25%). The toxicology analysis was carried out to determine the production of aflatoxins and the level present in the groundnut. This was carried out using a Romer Mycosep Column kit for aflatoxin to clean-up, and High-Performance Liquid Chromatography (HPLC) to detect the aflatoxins and determine the levels. Total aflatoxins were detected in the groundnuts, and they ranged between 0.36 ppb and 0.42 ppb. The levels of total aflatoxins were not beyond the regulatory standard of National Agency for Food and Drugs Administration and Control- NAFDAC (for ready to eat food, 4µg/kg while food that will still undergo processing 20µg/kg). Thus, the groundnut samples from the four markets are fit for consumption.

Evaluation of the Susceptibility of Lotus Seeds (Nelumbo nucifera Gaertn.) to Aspergillus flavus Infection and Aflatoxin Contamination.

The seeds of lotus (Nelumbo nucifera Gaertn.) have been used as significant medicinal and nutritional ingredients worldwide. The abundant proteins and polysaccharides in lotus seeds make them susceptible to contamination by aflatoxin (AF), a fungal toxic metabolite. This study was conducted to investigate the susceptibility of lotus seeds at different stages of ripening to AF contamination, as well as the mechanism of the contamination. Seven groups of lotus receptacles with seeds at different ripening stages (A-G, from immature to mature) were used for the experiment. Spores of Aspergillus flavus, an AF producer, were inoculated on the water-gap area of the seeds in each receptacle. Then, each receptacle was covered with a sterilized bag, and its stalk part was soaked in water containing a life-prolonging agent, after which it was kept at room temperature for 14 days. The AF content of each whole inoculated seed from the A-G groups and that of each seed part (pericarp, cotyledon, and embryo) from the D and E groups were determined using high-performance liquid chromatography. Microtome sections were prepared from the samples and observed under a light microscope and scanning electron microscope. The seeds from the A and D groups had higher AF contents than the seeds from the B, C, E, F, and G groups, indicating that the condition of the water-gap area and the development of the embryo and cotyledon parts of the seeds are associated with AF contamination.

Evaluation of antifungal activity of natural compounds on growth and aflatoxin B1 production of Aspergillus parasiticus and Aspergillus flavus.

Aspergillus species cause broad spectrum infections especially invasive lethal infections in immunocompromised patients. This study aimed to assess the antifungal activity of plants and compounds including Aloe vera, Thyme, carvacrol, and nano-encapsulation of carvacrol on the growth and production of aflatoxin B1 production by Aspergillus parasiticus and Aspergillus flavus. Minimum inhibitory concentrations of extracts Aloe vera, Thyme, carvacrol, and nanocarvacrol, and fluconazole as a control were determined according to Clinical and Laboratory Standards Institute by serial microdilution protocol. Then, the effect of inhibitory concentrations of these compounds on the aflatoxin B1 production level was evaluated by real-time PCR and high-performance liquid chromatography. Our results indicate that the Aspergillus parasiticus and Aspergillus flavusare sensitive to selected plants and compounds. Our findings showed that the compounds are appropriate alternative candidates against growth and production of aflatoxin of Aspergillus spp.

Whole genome sequencing and annotation of Aspergillus flavus JAM-JKB-B HA-GG20.

Groundnuts are mostly contaminated with the mold Aspergillus flavus which produces a carcinogenic mycotoxin called as aflatoxin. It is very important to understand the genetic factors underlying its pathogenicity, regulation, and biosynthesis of secondary metabolites and animal toxicities, but it still lacks useful information due to certain gaps in the era of modern technology. Therefore, the present study was considered to determine the key genes and metabolites involved in the biosynthesis of aflatoxin by using a molecular approach in a virulent strain of Aspergillus. The whole genome sequence of highly toxic and virulent Aspergillus isolates JAM-JKB-B HA-GG20 revealed 3,73,54,834bp genome size, 2, 26, 257 number of contigs with N50 value of 49,272bp, 12,400 genes and 48.1% of GC contained respectively. The genome sequence was compared with other known aflatoxin producing and non-producing genome of Aspergillus spp. and 61 secondary metabolite (SM) gene clusters were annotated with the toxic strain JAM-JKB-BHA-GG20 which showed similarity with other Aspergillus spp. A total number of eight genes (ver-1, AflR, pksA, uvm8, omt1, nor-1, Vha and aflP) were identified related to biosynthesis of aflatoxin and ochratoxin. Also, 69 SSR with forward and reverse primers and 137 di and tri nucleotide motifs were identified in the nucleotide sequence region related to aflatoxin gene pathway. The genes and putative metabolites identified in this study are potentially involved in host invasion and pathogenicity. As such, the genomic information obtained in this study is helpful in understanding aflatoxin gene producing pathway in comparison to other Aspergillus spp. and predicted presence of other secondary metabolites clusters viz. Nrps, T1pks etc. genes associated with a biosynthesis of OTA mycotoxin.

The potential use of ozone as antifungal and antiaflatoxigenic agent in nuts and its effect on nutritional quality.

Ozone gas is considered as a safe antimicrobial agent in food industries. Here, we evaluated the antifungal and antiaflatoxigenic activities of ozone against fungal contamination in nuts. The most predominant fungal genera in nuts were Aspergillus, Penicillium, Fusarium, and Rhizopus. Ozone (4 ppm) significantly reduced the fungal sporulation of A. flavus and their aflatoxin production. Interestingly, ozone treatment of nuts reduced the total fungal count and increased aflatoxins degradation by approximately 95% and 85%, respectively. Ozone displayed high efficiency to increase the permeability of cell membrane and injury of cell wall of fungi. Increasing the exposure time of ozone in nuts up to 180 minutes showed to reduce the total lipid, carbohydrates, and protein by around 41.2%, 42.7% and 38.4% respectively, in pistachio, almond and peanuts. In conclusion, ozonation is a suitable decontaminating approach for reducing the microbial load in nuts, when used with suitable exposure time.

Determination of Aflatoxins in Pig Feed. Using the Thin Layer Chromatography Technique

Aflatoxins are the most important mycotoxins in terms of food safety, due to their frequency and toxicity, they are characterized by being secondary metabolites synthesized by fungi. In order to determine the presence of aflatoxins in pork feed, five samples from different commercial companies were analyzed. The methodology used prior to the sample extraction process was the thin layer chromatography technique, observed using long-wave ultraviolet light. It could be seen in samples 1,3,4,5, the presence of Aflatoxins, toxicogenic strains of Aspergillus parasiticus, which produce aflatoxins, G1 and G2, is characterized by the fluorescence coloration, in sample 2 the presence was observed of aflatoxins toxicogenic strains of Aspergillus flavus. Toxigenic strains of Aspergillus flavus generally produce only aflatoxins B1 and B2, while toxicogenic strains of Aspergillus parasiticus produce aflatoxins B1, B2, G1 and G2. AFB1 aflatoxins have been considered obvious carcinogens in experimental animals. It is important to carry out systematic research with low-cost analytical techniques to understand the mechanisms of production and action of aflatoxins in foods, in order to prevent, monitor and control these types of toxins.