Characterization of a Novel Monoclonal Antibody with High Affinity and Specificity against Aflatoxins: A Discovery from Rosetta Antibody-Ligand Computational Simulation.

Aflatoxin B1 (AFB1) accumulates in crops, where it poses a threat to human health. To detect AFB1, anti-AFB1 monoclonal antibodies have been developed and are widely used. While the sensitivity and specificity of these antibodies have been extensively studied, information regarding the atomic-level docking of AFB1 (and its derivatives) with these antibodies is limited. Such information is crucial for understanding the key interactions that are required for high affinity and specificity in aflatoxin binding. First, a 3D comparative model of anti-AFB1 antibody (Ab-4B5G6) was predicted from the sequence using RosettaAntibody. We then utilized RosettaLigand to dock AFB1 onto ten homology models, producing a total of 10,000 binding modes. Interestingly, the best-scoring mode predicted strong interactions involving four sites within the heavy chain: ALA33, ASN52, HIS95, and TRP99. Importantly, these strong binding interactions exclusively involve the variable domain of the heavy chain. The best-scoring mode with AFB1 was also obtained through AF multimer combined with RosettaLigand, and two interactions at TRP and HIS were consistent with those found by Rosetta antibody-ligand computational simulation. The role of tryptophan in π interactions in antibodies was confirmed through mutation experiments, and the resulting mutant (W99A) exhibited a >1000-fold reduction in binding affinity for AFB1 and analogs, indicating the effect of tryptophan on the stability of CDR-H3 region. Additionally, we evaluated the binding of two glycolic acid-derived molecular derivatives (with impaired hydrogen bonding potential), and these derivatives (AFB2-GA and AFG2-GA) demonstrated a very weak binding affinity for Ab-4B5G6. The heavy chain was successfully isolated, and its sensitivity and specificity were consistent with those of the intact antibody. The homology models of variable heavy (VH) single-domain antibodies were established by RosettaAntibody, and the docking analysis revealed the same residues, including Ala, His, and Trp. Compared to the potential binding mode of fragment variable (FV) region, the results from a model of VH indicated that there are seven models involved in hydrophobic interaction with TYR32, which is usually referred to as polar amino acid and has both hydrophobic and hydrophilic features depending on the circumstances. Our work encompasses the entire process of Rosetta antibody-ligand computational simulation, highlighting the significance of variable heavy domain structural design in enhancing molecular interactions.

The occurrence and human health risk assessment of total and aflatoxin B1 in selected food commodities in Bhutan.

Aflatoxins are mycotoxins that contaminate staple foods globally and pose a significant health risk. To the best of our knowledge, information on the occurrence of aflatoxins in Bhutanese diets is scarce. This study aimed to estimate the aflatoxin levels in selected foodstuffs in Bhutan and determine the health risk associated with aflatoxin exposure. Ten different types of food commodities were randomly collected from farmers' markets, shelves of supermarkets, and wholesale and retail shops from 20 districts of the country. The samples were subjected to analysis by an enzyme-linked immunosorbent assay for both total aflatoxins (B1, B2, G1 and G2) and aflatoxin B1. Among the 315 samples included, 48.81% and 79.35% were positive for total aflatoxins and aflatoxin B1, respectively. The overall mean total aflatoxin concentration was 11.49 ± 12.83 µg/kg, and that for B1 was 17.62 ± 23.99 µg/kg. The most prevalent food commodity with the highest aflatoxin contamination was chili products. In addition, the estimated daily intake and margin of exposure to aflatoxin B1 via the consumption of chili products ranged from 0.98 to 5.34 ng kg-1 bw day-1 and from 74.90 to 408.10, indicating a risk for public health. The liver cancer risk was estimated to be 0.01 and 0.007 cancers per year per 100,000 population resulting from the consumption of chili products. The present findings revealed the presence of total aflatoxins and aflatoxin B1 in the selected samples. The margin of exposure values was exorbitant, demanding a stringent public health measure. Notably, these results suggest the need for routine monitoring of aflatoxin contamination in the region and stress rigorous safety management strategies to reduce exposure.

Antifungal, antiaflatoxigenic, and cytotoxic properties of bioactive secondary metabolites derived from Bacillus species.

Aflatoxins (AFs) are hazardous carcinogens and mutagens produced by some molds, particularly Aspergillus spp. Therefore, the purpose of this study was to isolate and identify endophytic bacteria, extract and characterize their bioactive metabolites, and evaluate their antifungal, antiaflatoxigenic, and cytotoxic efficacy against brine shrimp (Artemia salina) and hepatocellular carcinoma (HepG2). Among the 36 bacterial strains isolated, ten bacterial isolates showed high antifungal activity, and thus were identified using biochemical parameters and MALDI-TOF MS. Bioactive metabolites were extracted from two bacterial isolates, and studied for their antifungal activity. The bioactive metabolites (No. 4, and 5) extracted from Bacillus cereus DSM 31T DSM, exhibited strong antifungal capabilities, and generated volatile organic compounds (VOCs) and polyphenols. The major VOCs were butanoic acid, 2-methyl, and 9,12-Octadecadienoic acid (Z,Z) in extracts No. 4, and 5 respectively. Cinnamic acid and 3,4-dihydroxybenzoic acid were the most abundant phenolic acids in extracts No. 4, and 5 respectively. These bioactive metabolites had antifungal efficiency against A. flavus and caused morphological alterations in fungal conidiophores and conidiospores. Data also indicated that both extracts No. 4, and 5 reduced AFB1 production by 99.98%. On assessing the toxicity of bioactive metabolites on A. salina the IC50 recorded 275 and 300 µg/mL, for extracts No. 4, and 5 respectively. Meanwhile, the effect of these extracts on HepG2 revealed that the IC50 of extract No. 5 recorded 79.4 µg/mL, whereas No. 4 showed no cytotoxic activity. It could be concluded that bioactive metabolites derived from Bacillus species showed antifungal and anti-aflatoxigenic activities, indicating their potential use in food safety.

A miniaturized QuEChERS and UPLC-MS/MS method for the determination of mycotoxins in cashew nuts.

This study aimed to develop and validate a multi-mycotoxin analysis method applied to cashew nuts by employing a miniaturized QuEChERS method followed by determination by ultra-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). Satisfactory recoveries for the concentrations 1, 10 and 30 ng g-1, ranging from 66% (fumonisin B1) to 110% (ochratoxin A) and relative standard deviations lower than 9% (fumonisin B2) were obtained for the target compounds. Limits of quantification ranged from 0.004 ng g-1 (sterigmatocystin) to 0.59 ng g-1 (alternariol). The applicability of the analytical method was verified by analyzing 30 cashew nut samples from the city of Rio de Janeiro, RJ, southeastern Brazil. Aflatoxins M1, G2, G1, B2, B1, ochratoxin A and sterigmatocystin were detected, respectively, in 27%, 10%, 17%, 30%, 30%, 30% and 50% of the analyzed samples, at maximum concentrations of 0.56, 0.67, 1.43, 2.02, 4.93, 4.81, and 0.35 ng g-1. The maximum limit established by Brazilian legislation for aflatoxins was not exceeded by any of the analyzed samples.

Increased Dissemination of Aflatoxin- and Zearalenone-Producing Aspergillus spp. and Fusarium spp. during Wet Season via Houseflies on Dairy Farms in Aguascalientes, Mexico.

Crops contamination with aflatoxins (AFs) and zearalenone (ZEA) threaten human and animal health; these mycotoxins are produced by several species of Aspergillus and Fusarium. The objective was to evaluate under field conditions the influence of the wet season on the dissemination of AF- and ZEA-producing fungi via houseflies collected from dairy farms. Ten dairy farms distributed in the semi-arid Central Mexican Plateau were selected. Flies were collected in wet and dry seasons at seven points on each farm using entomological traps. Fungi were isolated from fly carcasses via direct seeding with serial dilutions and wet chamber methods. The production of AFs and ZEA from pure isolates was quantified using indirect competitive ELISA. A total of 693 Aspergillus spp. and 1274 Fusarium spp. isolates were obtained, of which 58.6% produced AFs and 50.0% produced ZEA (491 ± 122; 2521 ± 1295 µg/kg). Houseflies and both fungal genera were invariably present, but compared to the dry season, there was a higher abundance of flies as well as AF- and ZEA-producing fungi in the wet season (p < 0.001; 45.3/231 flies/trap; 8.6/29.6% contaminated flies). These results suggest that rainy-weather conditions on dairy farms increase the spread of AF- and ZEA-producing Aspergillus spp. and Fusarium spp. through houseflies and the incorporation of their mycotoxins into the food chain.

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.

Effects of Different Shelling Methods on Data Variability during Field Screening for Reduced Aflatoxin Contamination in Maize.

Non-genetic variation limits the identification of novel maize germplasm with genetic markers for reduced Aspergillus flavus infection and aflatoxin contamination. Aflatoxin measurements can vary substantially within fields containing the same germplasm following inoculation with A. flavus. While some variation is expected due to microenvironmental differences, components of field screening methodologies may also contribute to variability in collected data. Therefore, the objective of this study is to test the effects of three different shelling methods (whole ear (WE), ear end removal (EER), and inoculation site-surrounding (ISS)) to obtain bulk samples from maize on aflatoxin measurements. Five ears per row of three inbred lines and two hybrids were inoculated with A. flavus, then shelled using the three different methods, and aflatoxin was quantified. Overall, EER and ISS resulted in reduced coefficients of variance (CVs) in comparison to WE for both inbred and hybrid maize lines, with two exceptions. Susceptible B73 showed increased CVs with both EER and ISS compared to WE, and resistant Mp719's EER CVs marginally increased compared to WE. While WE is the standard practice for most breeding programs due to its technical simplicity, EER and ISS may allow for finely phenotyping parental lines for further breeding applications.

Enhanced detection of Aspergillus flavus in peanut kernels using a multi-scale attention transformer (MSAT): Advancements in food safety and contamination analysis.

In this study, a multi-scale attention transformer (MSAT) was coupled with hyperspectral imaging for classifying peanut kernels contaminated with diverse Aspergillus flavus fungi. The results underscored that the MSAT significantly outperformed classic deep learning models, due to its sophisticated multi-scale attention mechanism which enhanced its classification capabilities. The multi-scale attention mechanism was utilized by employing several multi-head attention layers to focus on both fine-scale and broad-scale features. It also integrated a series of scale processing layers to capture features at different resolutions and incorporated a self-attention mechanism to integrate information across different levels. The MSAT model achieved outstanding performance in different classification tasks, particularly in distinguishing healthy peanut kernels from those contaminated with aflatoxigenic fungi, with test accuracy achieving 98.42±0.22%. However, it faced challenges in differentiating peanut kernels contaminated with aflatoxigenic fungi from those with non-aflatoxigenic contamination. Visualization of attention weights explicitly revealed that the MSAT model's multi-scale attention mechanism progressively refined its focus from broad spatial-spectral features to more specialized signatures. Overall, the MSAT model's advanced processing capabilities marked a notable advancement in the field of food quality safety, offering a robust and reliable tool for the rapid and accurate detection of Aspergillus flavus contaminations in food.

The target of rapamycin signaling pathway regulates vegetative development, aflatoxin biosynthesis, and pathogenicity in Aspergillus flavus.

The target of rapamycin (TOR) signaling pathway is highly conserved and plays a crucial role in diverse biological processes in eukaryotes. Despite its significance, the underlying mechanism of the TOR pathway in Aspergillus flavus remains elusive. In this study, we comprehensively analyzed the TOR signaling pathway in A. flavus by identifying and characterizing nine genes that encode distinct components of this pathway. The FK506-binding protein Fkbp3 and its lysine succinylation are important for aflatoxin production and rapamycin resistance. The TorA kinase plays a pivotal role in the regulation of growth, spore production, aflatoxin biosynthesis, and responses to rapamycin and cell membrane stress. As a significant downstream effector molecule of the TorA kinase, the Sch9 kinase regulates aflatoxin B1 (AFB1) synthesis, osmotic and calcium stress response in A. flavus, and this regulation is mediated through its S_TKc, S_TK_X domains, and the ATP-binding site at K340. We also showed that the Sch9 kinase may have a regulatory impact on the high osmolarity glycerol (HOG) signaling pathway. TapA and TipA, the other downstream components of the TorA kinase, play a significant role in regulating cell wall stress response in A. flavus. Moreover, the members of the TapA-phosphatase complexes, SitA and Ppg1, are important for various biological processes in A. flavus, including vegetative growth, sclerotia formation, AFB1 biosynthesis, and pathogenicity. We also demonstrated that SitA and Ppg1 are involved in regulating lipid droplets (LDs) biogenesis and cell wall integrity (CWI) signaling pathways. In addition, another phosphatase complex, Nem1/Spo7, plays critical roles in hyphal development, conidiation, aflatoxin production, and LDs biogenesis. Collectively, our study has provided important insight into the regulatory network of the TOR signaling pathway and has elucidated the underlying molecular mechanisms of aflatoxin biosynthesis in A. flavus.

Analysis and evaluation of in vitro bioaccessibility of aflatoxins B1, B2, G1 and G2 in plant-based milks.

Plant-based milks emerge as a healthy and vegan alternative for human diet, but these foodstuffs are susceptible to be contaminated by aflatoxins. A new method based on SPE and HPLC-MS/MS analysis was optimized and validated to test the presence of aflatoxins B1, B2, G1 and G2 analysis in almond, oat, rice and soy commercial milks. Moreover, aflatoxin bioaccessibility was evaluated through an in vitro digestion assay applied to each type of spiked milk. Aflatoxins B1, B2 and G1 were detected in one soy milk sample below the LOQ, fulfilling the limits stablished by the European Legislation. The final bioaccessibility percentages were highly dependent on the type of mycotoxin and sample matrix, the highest and the lowest values were obtained for AFB2 (82%-92%) and AFG1 (15%-30%), whereas AFB1 (28%-50%) and AFG2 (32%-76%) values resulted more influenced by the plant-based milk matrix.

Study on the metabolic changes and regulatory mechanism of Aspergillus flavus conidia germination.

Aspergillus flavus conidia are widespread in air; they attach to food and feed crops and secrete aflatoxins, which results in serious contamination. Germination of A. flavus conidia is the most critical step in contamination of food by A. flavus. This study aims to gain an insight into A. flavus conidia through dormancy to germination to provide a theoretical basis for inhibition of A. flavus conidia germination. The morphological changes and regulation mechanism of A. flavus conidia germination at 0, 4, 8, and 12 hours were observed. Transcriptomic and metabolomic analyses showed that conidia became active from dormancy (0 hour) to the initial stage of germination (4 hours), cellular respiration and energy metabolism increased, and amino acids and lipids were synthesized rapidly. The number of differentially expressed genes and differential metabolites was highest at this stage. Besides, we found that conidia germination had selectivity for different carbon and nitrogen sources. Compared with monosaccharides, disaccharides, as the only carbon source, significantly promoted the germination of conidia. Moreover, MepA, one of genes in the ammonium transporter family was studied. The gene deletion mutant ΔMepA had a significant growth defect, and the expression of MeaA was significantly upregulated in ΔMepA compared with the wild-type, indicating that both MepA and MeaA played an important role in transporting ammonium ions.IMPORTANCEThis is the first study to use combined transcriptomic and metabolomics analyses to explore the biological changes during germination of Aspergillus flavus conidia. The biological process with the highest changes occurred in 0-4 hours at the initial stage of germination. Compared with polysaccharides, monosaccharides significantly increased the size of conidia, while significantly decreasing the germination rate of conidia. Both MeaA and MepA were involved in ammonia transport and metabolism during conidia germination.

Phytochemical-mediated regulation of aflatoxigenic fungi contamination in a shifting climate and environment.

Mycotoxin contamination poses a significant problem in developing countries, particularly in northern Pakistan's fluctuating climate. This study aimed to assess aflatoxin contamination in medicinal and condiment plants in Upper Dir (dry-temperate) and Upper Swat (moist-temperate) districts. Plant samples were collected and screened for mycotoxins (Aflatoxin-B1 and Aflatoxin-B-2). Results showed high levels of AFB-1 (11,505.42 ± 188.82) as compared to AFB-2 (846 ± 241.56). The maximum contamination of AFB-1 in Coriandrum sativum (1154.5 ± 13.43 ng to 3328 ± 9.9 ng) followed by F. vulgare (883 ± 9.89 ng to 2483 ± 8.4 ng), T. ammi (815 ± 11.31 ng to 2316 ± 7.1 ng), and C. longa (935.5 ± 2.12 ng to 2009 ± 4.2 ng) while the minimum was reported in C. cyminum (671 ± 9.91 ng to 1995 ± 5.7 ng). Antifungal tests indicated potential resistance in certain plant species (C. cyminum) while A. flavus as the most toxins contributing species due to high resistance below 80% (54.2 ± 0.55 to 79.5 ± 2.02). HPLC analysis revealed hydroxyl benzoic acid (5136 amu) as the dominant average phytochemical followed by phloroglucinol (4144.31 amu) with individual contribution of 8542.08 amu and 12,181.5 amu from C. cyaminum. The comparison of average phytochemicals revealed the maximum concentration in C. cyminum (2885.95) followed by C. longa (1892.73). The findings revealed a statistically significant and robust negative correlation (y = - 2.7239 × + 5141.9; r = - 0.8136; p < 0.05) between average mycotoxins and phytochemical concentrations. Temperature positively correlated with aflatoxin levels (p < 0.01), while humidity had a weaker correlation. Elevation showed a negative correlation (p < 0.05), while geographical factors (latitude and longitude) had mixed correlations (p < 0.05). Specific regions exhibited increasing aflatoxin trends due to climatic and geographic factors.

Raman Hyperspectral Imaging as a Potential Tool for Rapid and Nondestructive Identification of Aflatoxin Contamination in Corn Kernels.

The potential of Raman hyperspectral imaging with a 785 nm excitation line laser was examined for the detection of aflatoxin contamination in corn kernels. Nine-hundred kernels were artificially inoculated in the laboratory, with 300 kernels each inoculated with AF13 (aflatoxigenic) fungus, AF36 (nonaflatoxigenic) fungus, and sterile distilled water (control). One-hundred kernels from each treatment were subsequently incubated for 3, 5, and 8 days. The mean spectra of single kernels were extracted from the endosperm side and the embryo area of the germ side, and local Raman peaks were identified based upon the calculated reference spectra of aflatoxin-negative and -positive categories separately. The principal component analysis-linear discriminant analysis models were established using different types of variable inputs including original full spectra, preprocessed full spectra, and identified local peaks over kernel endosperm-side, germ-side, and both sides. The results of the established discriminant models showed that the germ-side spectra performed better than the endosperm-side spectra. Based upon the 20 ppb-threshold, the best mean prediction accuracy of 82.6% was achieved for the aflatoxin-negative category using the original spectra in the combined form of both kernel sides, and the best mean prediction accuracy of 86.7% was obtained for the -positive category using the preprocessed germ-side spectra. Based upon the 100 ppb-threshold, the best mean prediction accuracies of 85.0% and 89.6% were achieved for the aflatoxin-negative and -positive categories separately, using the same type of variable inputs for the 20 ppb-threshold. In terms of overall prediction accuracy, the models established upon the original spectra in the combined form of both kernel sides achieved the best predictive performance, regardless of the threshold. The mean overall prediction accuracies of 81.8% and 84.5% were achieved with the 20 ppb- and 100 ppb-thresholds, respectively.

Peanut Rhizosphere Achromobacter xylosoxidans Inhibits Aspergillus flavus Development and Aflatoxin Synthesis by Inducing Apoptosis through Targeting the Cell Membrane.

Contamination of crop seeds and feed with Aspergillus flavus and its associated aflatoxins presents a significant threat to human and animal health due to their hepatotoxic and carcinogenic properties. To address this challenge, researchers have screened for potential biological control agents in peanut soil and pods. This study identified a promising candidate, a strain of the nonpigmented bacterium, Achromobacter xylosoxidans ZJS2-1, isolated from the peanut rhizosphere in Zhejiang Province, China, exhibiting notable antifungal and antiaflatoxin activities. Further investigations demonstrated that ZJS2-1 active substances (ZAS) effectively inhibited growth at a MIC of 60 µL/mL and nearly suppressed AFB1 production by 99%. Metabolomic analysis revealed that ZAS significantly affected metabolites involved in cell wall and membrane biosynthesis, leading to compromised cellular integrity and induced apoptosis in A. flavus through the release of cytochrome c. Notably, ZAS targeted SrbA, a key transcription factor involved in ergosterol biosynthesis and cell membrane integrity, highlighting its crucial role in ZJS2-1's biocontrol mechanism. Moreover, infection of crop seeds and plant wilt caused by A. flavus can be efficiently alleviated by ZAS. Additionally, ZJS2-1 and ZAS demonstrated significant inhibitory effects on various Aspergillus species, with inhibition rates ranging from 80 to 99%. These findings highlight the potential of ZJS2-1 as a biocontrol agent against Aspergillus species, offering a promising solution to enhance food safety and protect human health.

Molecular and aflatoxigenicity analyses of Aspergillus flavus isolates indigenous to grain corn in Malaysia; potentials for biological control.

AIMS: The present work aimed to distinguish the indigenous Aspergillus flavus isolates obtained from the first (pioneer) grain corn farms in Terengganu, Malaysia, into aflatoxigenic and non-aflatoxigenic by molecular and aflatoxigenicity analyses, and determine the antagonistic capability of the non-aflatoxigenic isolates against aflatoxigenic counterparts and their aflatoxin production in vitro. METHODS AND RESULTS: Seven A. flavus isolates previously obtained from the farms were characterized molecularly and chemically. All isolates were examined for the presence of seven aflatoxin biosynthesis genes, and their aflatoxigenicity was confirmed using high performance liquid chromatography with fluorescence detector. Phylogenetic relationships of all isolates were tested using ITS and ß-tubulin genes. Of the seven isolates, two were non-aflatoxigenic, while the remaining were aflatoxigenic based on the presence of all aflatoxin biosynthesis genes tested and the productions of aflatoxins B1 and B2. All isolates were also confirmed as A. flavus following phylogenetic analysis. The indigenous non-aflatoxigenic isolates were further examined for their antagonistic potential against aflatoxigenic isolates on 3% grain corn agar. Both non-aflatoxigenic isolates significantly reduced AFB1 production of the aflatoxigenic isolates. CONCLUSION: The indigenous non-aflatoxigenic A. flavus strains identified in the present work were effective in controlling the aflatoxin production by the aflatoxigenic A. flavus isolates in vitro and can be utilized for in situ testing.

A smartphone-enabled visual platform for detecting aflatoxins in peanut oil using colorimetric analysis coupled with magnetic imprinted solid-phase extraction.

The study aimed to develop a rapid and sensitive colorimetric platform based on the Emerson reaction to visualize and determine total aflatoxins (AFs) in peanut oil. This method offers the advantage of fast screening for AFs (AFB1, AFB2, AFG1, and AFG2), eliminating the need for specific antibodies. The proposed approach combined colorimetric detection with magnetic dummy imprinted solid-phase extraction and purification, enhancing sensitivity and selectivity. The oxidizer aided the colorless AFs in reacting with 4-aminoantipyrine, producing green condensates. Thus, a dual-mode approach was developed for AFs detection, employing both UV-vis colorimetric and smartphone-based colorimetry. Both methods showed a good linear relationship with the concentration of AFs. Notably, the smartphone-based method demonstrated a detection range of 0.5-57 µg/kg, with a detection limit as low as 0.21 µg/kg. The suggested colorimetric methods present a promising potential for onsite detection and fast screening of AFs in actual samples.

Mycobiota contaminating some market cake samples with reference to their toxin and enzyme.

Fungi can spoil the majority of baked products. Spoilage of cake during storage is commonly associated with fungi. Therefore, this study aimed to assess the quality of different types of cakes sold in the market. The most predominant fungal genera in the tested cake samples (14 samples) were Aspergillus spp., and Penicillium spp. On Potato Dextrose Agar (PDA), the medium fungal total count was 43.3 colonies /g. Aspergillus was the most dominant genus and was isolated from six samples of cake. Aspergillus was represented by 3 species namely, A. flavus, A. niger, and A. nidulans, represented by 13.32, 19.99, and 3.33 colonies /g respectively. On Malt Extract Agar (MEA) Medium, the fungal total count was 123.24 colonies / g. Aspergillus was the most dominant isolated genus from 11 samples of cake and was represented by 5 species, namely, A. flavus, A. niger, A. ochraceous, A. terreus, and A. versicolor (26. 65, 63.29, 3.33, 6.66, and 3.33 colonies / g , respectively). Twenty-four isolates (88.88 %) of the total tested twenty-seven filamentous fungi showed positive results for amylase production. Ten isolates (37.03%) of the total tested filamentous fungi showed positive results for lipase production, and finally eleven isolates (40.74 %) of the total fungal isolates showed positive results for protease production. Aflatoxins B1, B2, G1, G2, and ochratoxin A were not detected in fourteen collected samples of cake. In this study, clove oil was the best choice overpeppermint oil and olive oil for preventing mold development when natural agents were compared. It might be due to the presence of a varietyof bioactive chemical compounds in clove oil, whose major bioactive component is eugenol, which acts as an antifungal reagent. Therefore, freshly baked cake should be consumed within afew days to avoid individuals experiencing foodborne illnesses.

Aflatoxins in the rice production chain: A review on prevalence, detection, and decontamination strategies.

Rice (Oryza sativa L.) is one of the most consumed cereals that along with several important nutritional constituents typically provide more than 21% of the caloric requirements of human beings. Aflatoxins (AFs) are toxic secondary metabolites of several Aspergillus species that are prevalent in cereals, including rice. This review provides a comprehensive overview on production factors, prevalence, regulations, detection methods, and decontamination strategies for AFs in the rice production chain. The prevalence of AFs in rice is more prominent in African and Asian than in European countries. Developed nations have more stringent regulations for AFs in rice than in the developing world. The contamination level of AFs in the rice varied at different stages of rice production chain and is affected by production practices, environmental conditions comprising temperature, humidity, moisture, and water activity as well as milling operations such as de-husking, parboiling, and polishing. A range of methods including chromatographic techniques, immunochemical methods, and spectrophotometric methods have been developed, and used for monitoring AFs in rice. Chromatographic methods are the most used methods of AFs detection followed by immunochemical techniques. AFs decontamination strategies adopted worldwide involve various physical, chemical, and biological strategies, and even using plant materials. In conclusion, adopting good agricultural practices, implementing efficient AFs detection methods, and developing innovative aflatoxin decontamination strategies are imperative to ensure the safety and quality of rice for consumers.

Aflatoxin biosynthesis regulators AflR and AflS: DNA binding affinity, stoichiometry, and kinetics.

Aflatoxins (AFs), potent foodborne carcinogens produced by Aspergillus fungi, pose significant health risks worldwide and present challenges to food safety and productivity in the food chain. Novel strategies for disrupting AF production, cultivating resilient crops, and detecting contaminated food are urgently needed. Understanding the regulatory mechanisms of AF production is pivotal for targeted interventions to mitigate toxin accumulation in food and feed. The gene cluster responsible for AF biosynthesis encodes biosynthetic enzymes and pathway-specific regulators, notably AflR and AflS. While AflR, a DNA-binding protein, activates gene transcription within the cluster, AflS enhances AF production through mechanisms that are not fully understood. In this study, we developed protocols to purify recombinant AflR and AflS proteins and utilized multiple assays to characterize their interactions with DNA. Our biophysical analysis indicated that AflR and AflS form a complex. AflS exhibited no DNA-binding capability on its own but unexpectedly reduced the DNA-binding affinity of AflR. Additionally, we found that AflR achieves its binding specificity through a mechanism in which either two copies of AflR or its complex with AflS bind to target sites on DNA in a highly cooperative manner. The estimated values of the interaction parameters of AflR, AflS and DNA target sites constitute a fundamental framework against which the function and mechanisms of other AF biosynthesis regulators can be compared.

Self-assembled thymol-betaine co-crystals with controlled release and hygroscopic properties as green preservatives for aflatoxin prevention.

Mycotoxins are representative contaminants causing food losses and food safety problems worldwide. Thymol can effectively inhibit pathogen infestation and aflatoxin accumulation during grain storage, but high volatility limits its application. Here, a thymol-betaine co-crystal system was synthesized through grinding-induced self-assembly. The THY-TMG co-crystal exhibited excellent thermal stability with melting point of 91.2 °C owing to abundant intermolecular interactions. Remarkably, after 15 days at 30 °C, the release rate of thymol from co-crystal was only 55%, far surpassing that of pure thymol. Notably, the co-crystal demonstrated the ability to bind H2O in the environment while controlling the release of thymol, essentially acting as a desiccant. Moreover, the co-crystals effectively inhibited the growth of Aspergillus flavus and the biosynthesis of aflatoxin B1. In practical terms, the THY-TMG co-crystal was successful in preventing AFB1 contamination and nutrients loss in peanuts, thereby prolonging their shelf-life under conditions of 28 °C and 70% RH.