Complexity of fungal polyketide biosynthesis and function.

Where does one draw the line between primary and secondary metabolism? The answer depends on the perspective. Microbial secondary metabolites (SMs) were at first believed not to be very important for the producers because they are dispensable for growth under laboratory conditions. However, such compounds become important in natural niches of the organisms, and some are of prime importance for humanity. Polyketides are an important group of SMs with aflatoxin as a well-known and well-characterized example. In Aspergillus spp., all 34 afl genes encoding the enzymes for aflatoxin biosynthesis are located in close vicinity on chromosome III in a so-called gene cluster. This led to the assumption that most genes required for polyketide biosynthesis are organized in gene clusters. Recent research, however, revealed an enormous complexity of the biosynthesis of different polyketides, ranging from individual polyketide synthases to a gene cluster producing several compounds, or to several clusters with additional genes scattered in the genome for the production of one compound. Research of the last decade furthermore revealed a huge potential for SM biosynthesis hidden in fungal genomes, and methods were developed to wake up such sleeping genes. The analysis of organismic interactions starts to reveal some of the ecological functions of polyketides for the producing fungi.

Glucose and HODEs regulate Aspergillus ochraceus quorum sensing through the GprC-AcyA pathway.

Aspergillus ochraceus is the traditional ochratoxin A (OTA)-producing fungus with density-dependent behaviors, which is known as quorum sensing (QS) that is mediated by signaling molecules. Individual cells trend to adapt environmental changes in a "whole" flora through communications, allowing fungus to occupy an important ecological niche. Signals perception, transmission, and feedback are all rely on a signal network that constituted by membrane receptors and intracellular effectors. However, the interference of density information in signal transduction, which regulates most life activities of Aspergillus, have yet to be elucidated. Here we show that the G protein-coupled receptor (GPCR) to cAMP pathway is responsible for transmitting density information, and regulates the key point in life cycle of A. ochraceus. Firstly, the quorum sensing phenomenon of A. ochraceus is confirmed, and identified the density threshold is 103 spores/mL, which represents the low density that produces the most OTA in a series quorum density. Moreover, the GprC that classified as sugar sensor, and intracellular adenylate cyclase (AcyA)-cAMP-PKA pathway that in response to ligands glucose and HODEs are verified. Furthermore, GprC and AcyA regulate the primary metabolism as well as secondary metabolism, and further affects the growth of A. ochraceus during the entire life cycle. These studies highlight a crucial G protein signaling pathway for cell communication that is mediated by carbohydrate and oxylipins, and clarified a comprehensive effect of fungal development, which include the direct gene regulation and indirect substrate or energy supply. Our work revealed more signal molecules that mediated density information and connected effects on important adaptive behaviors of Aspergillus ochraceus, hoping to achieve comprehensive prevention and control of mycotoxin pollution from interrupting cell communication.

Detoxifying bacterial genes for deoxynivalenol epimerization confer durable resistance to Fusarium head blight in wheat.

Fusarium head blight (FHB) and the presence of mycotoxin deoxynivalenol (DON) pose serious threats to wheat production and food safety worldwide. DON, as a virulence factor, is crucial for the spread of FHB pathogens on plants. However, germplasm resources that are naturally resistant to DON and DON-producing FHB pathogens are inadequate in plants. Here, detoxifying bacteria genes responsible for DON epimerization were used to enhance the resistance of wheat to mycotoxin DON and FHB pathogens. We characterized the complete pathway and molecular basis leading to the thorough detoxification of DON via epimerization through two sequential reactions in the detoxifying bacterium Devosia sp. D6-9. Epimerization efficiently eliminates the phytotoxicity of DON and neutralizes the effects of DON as a virulence factor. Notably, co-expressing of the genes encoding quinoprotein dehydrogenase (QDDH) for DON oxidation in the first reaction step, and aldo-keto reductase AKR13B2 for 3-keto-DON reduction in the second reaction step significantly reduced the accumulation of DON as virulence factor in wheat after the infection of pathogenic Fusarium, and accordingly conferred increased disease resistance to FHB by restricting the spread of pathogenic Fusarium in the transgenic plants. Stable and improved resistance was observed in greenhouse and field conditions over multiple generations. This successful approach presents a promising avenue for enhancing FHB resistance in crops and reducing mycotoxin contents in grains through detoxification of the virulence factor DON by exogenous resistance genes from microbes.

Nursery pigs fed with feed contaminated by aflatoxin B1 (Aspergillus flavus) and anti-mycotoxin blend: Pathogenesis and negative impact on animal health and weight gain.

The present study aimed to evaluate whether a moderate dose of aflatoxin B1 in pigs' diet interferes with pigs' growth and health in the nursery phase and whether an anti-mycotoxin mixture minimizes the adverse effects of the toxin. One blend with Saccharomyces cerevisiae lysate, zeolite, silicon dioxide, propylene glycol, Carduus marianus extract, soy lecithin, and carbonate was used as an anti-mycotoxin. Four treatments, with six repetitions per treatment and three pigs/pen: Afla0-AntiMyc0 - negative control (without aflatoxin); Afla500-AntiMyc0 - positive control (500 ppb of aflatoxin); Afla0-AntiMyc1000 - 1000 mg/kg of anti-mycotoxin blend; Afla500-AntiMyc1000 - 500 ppb aflatoxin +1000 mg/kg of anti-mycotoxin blend. It was observed that pigs in the positive control (Afla500-AntiMyc0) had lower body weight and weight gain when compared to the other treatments during the experimental period. Also, pigs from Afla500-AntiMyc0 had lower feed intake between days 1-20 and 1 to 30 than Afla0-AntiMyc0. The pigs from Afla500-AntiMyc0 had higher levels of liver enzymes aspartate aminotransferase and alanine aminotransferase compared to other treatments. The pigs from Afla500-AntiMyc0 had higher villus height than the other treatments, while the folded size was smaller in this treatment. Crypts were deeper in the intestines of pigs in both treatments that consumed aflatoxin. In general, it is concluded that the intake of aflatoxin B1 by nursery pigs has negative impacts on the health and, consequently, the animals' growth performance; however, the addition of the contaminated feed with an anti-mycotoxin blend was able to protect the pigs, minimizing the adverse effects caused by the mycotoxin.

Impacts of intake of trichothecenes (Fusarium sporotrichioides) for dairy calves: Effects on animal growth, oxidative and inflammatory response.

The objective of the present study was to evaluate the impacts of trichothecenes (Fusarium sporotrichioides) for dairy calves on animal growth, oxidative and inflammatory responses in the presence or absence of essential oils. Twelve calves weaned at 70 days of age were divided into 2 groups: T-C (control) and T-EO (essential oils - oregano, thyme, basil and rosemary) in the period of 40 days consuming ration contaminated by trichothecenes (500 ppb). The animals in the T-EO group received a mixture of EOs via feed at a dosage of 0.75 mL per/kg of feed. Blood collections were performed on days 1, 20 and 40 for hematological and biochemical analyses; the fecal score was performed every 2 days on a scale of 1-5 and clinical examinations were performed 3 times during the experiment period. The animals were weighed at the beginning and at the end of the experiment; euthanasia of two calves per group for macroscopic and microscopic evaluation of several tissues (spleen, liver, duodenum, jejunum, ilium, cecum and colon) was performed at the end of the experiment. The calves in the T-EO group had a tendency (P = 0.07) of higher body weight when compared to the T-C. Treatment effect and treatment vs day interaction was detected for leukocytes and granulocytes variables, demonstrating a higher count of these cells in the T-EO group on both days (20 and 40), and the same behavior occurred for the distribution amplitude of erythrocytes (RDW). The enzymes alanine transferase (ALT), aspartate transferase (AST) and gamma glutamyl-transferase (GGT) showed higher serum activity in the T-C group (days 20 and 40). The levels of thiobarbituric acid reactive substances (TBARS) were lower in the serum of animals in the T-EO group. For calves in the T-EO group, glutathione S-transferase activity was higher in serum. Haptoglobulin and C-reactive protein levels were lower on days 20 and 40 in T-EO animals when compared to the T-C group. In the macroscopic and microscopic evaluations, which were collected at the end of the experiment after slaughtering the animals, liver and intestine did not show changes for the animals in the T-EO group, unlike the animals in the T-C group, which had moderately firm diffuse consistency of the liver and edema in the mesentery, as well as oxidative stress in tissues (liver, duodenum, jejunum, ileum, cecum and colon). The results concluded that the consumption of a mixture of EOs (essential oils - oregano, thyme, basil and rosemary) minimized the negative effects caused by trichothecenes in dairy calves, thus being an alternative to improving the immunological and antioxidant condition, as well as a possible adsorbent alternative.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples.

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Biocontrol Agents of Fusarium Head Blight in Wheat: A Meta-Analytic Approach to Elucidate Their Strengths and Weaknesses.

The use of biocontrol agents (BCAs) coping with fungal pathogens causing Fusarium head blight (FHB) is a compelling strategy for disease management, but a better elucidation of their effectiveness is crucial. Meta-analysis is the analysis of the results of multiple studies, which is typically performed to synthesize evidence from many possible sources in a formal probabilistic manner. This meta-analytic study, including 30 pathometric, biometric, physiochemical, genetic, and mycotoxin response variables reported in 56 studies, evidences the BCA effects on FHB in wheat. The effectiveness of BCAs of FHB in wheat in terms of pathogen abundance and disease reductions, biomass and yield conservation, and mycotoxin prevention/control was confirmed. BCAs showed higher efficacy (i) in studies published more recently; (ii) under controlled conditions; (iii) in high susceptible wheat cultivars; (iv) when Fusarium inoculation and BCA treatment did not occur directly on the plant (i.e., at the seed and kernel levels) in terms of disease development and mycotoxin control, and vice versa in terms of biomass conservation; (v) if Fusarium inoculation and BCA treatment occurred by spraying spikes in terms of yield; (vi) at 15 to 21 days post Fusarium inoculation or BCA treatment; and (vii) if they were filamentous fungi. However, BCAs overall were less efficacious than conventional agrochemicals, especially in terms of pathogen abundance and FHB reductions, as well as of mycotoxin prevention/control, although inconsistencies were reported among the investigated moderator variables. This study also highlights the complexity of reaching a good balance among BCA effects, and the need for further research.

High-throughput extraction and automatic purification of alternariol from edible and medicinal herbs based on aptamer-functionalized magnetic nanoparticles.

Mycotoxin contamination is widespread in plants and herbs, posing serious threats to the consumer and human health. Of them, alternariol (AOH) has attracted great attention as an "emerging" mycotoxin in medicinal herbs. However, a specific and high-throughput extraction method for AOH is currently lacking. Thus, developing an efficient pre-treatment technique for AOH detection is extremely vital. Here, a novel automated magnetic solid-phase extraction method was proposed for the highly efficient extraction of AOH. Combining the aptamer-functionalized magnetic nanoparticles (AMNPs) and the automatic purification instrument, AOH could be extracted in medicinal herbs in high throughput (20 samples) and a short time (30 min). The main parameters affecting extraction were optimized, and the method was finally carried out by incubation AMNPs with 3 mL of sample solution for 10 min, and then desorption in 75% methanol for liquid-phase detection. Under optimal conditions, good reproducibility, stability, and selectivity were realized with an adsorption capacity of 550.84 ng/mg. AOH extraction in three edible herbs showed good resistance to matrix interference with recovery rates from 86% to 111%. In combination with AMNPs and the automatic purification instrument, high-throughput and labor-free extraction of AOH in different complex matrices was achieved, which could be extended in other complex matrices.

Central role of Sigma-1 receptor in ochratoxin A-induced ferroptosis.

Ochratoxin A (OTA), a secondary fungal metabolite known for its nephrotoxic effects, is prevalent in various feeds and food items. Our recent study suggests that OTA-induced nephrotoxicity is linked to the Sigma-1 receptor (Sig-1R)-mediated mitochondrial pathway apoptosis in human proximal tubule epithelial-originated kidney-2 (HK-2) cells. However, the contribution of Sig-1R to OTA-induced nephrotoxicity involving other forms of regulated cell death, such as ferroptosis, remains unexplored. In this investigation, cell viability, malondialdehyde (MDA) levels, glutathione (GSH) levels, and protein expressions in HK-2 cells treated with OTA and/or Ferrostatin-1/blarcamesine hydrochloride/BD1063 dihydrochloride were assessed. The results indicate that a 24 h-treatment with 1 µM OTA significantly induces ferroptosis by inhibiting Sig-1R, subsequently promoting nuclear receptor coactivator 4 (NCOA4), long-chain fatty acid-CoA ligase 4 (ACSL4), arachidonate 5-lipoxygenase (ALOX5), autophagy protein 5 (ATG5), and ATG7, inhibiting ferritin heavy chain (FTH1), solute carrier family 7 member 11 (SLC7A11/xCT), glutathione peroxidase 4 (GPX4), peroxiredoxin 6 (PRDX6), and ferroptosis suppressor protein 1 (FSP1), reducing GSH levels, and increasing MDA levels (P < 0.05). In conclusion, OTA induces ferroptosis by inhibiting Sig-1R, subsequently promoting ferritinophagy, inhibiting GPX4/FSP1 antioxidant systems, reducing GSH levels, and ultimately increasing lipid peroxidation levels in vitro.

The characteristics, occurrence, and toxicological effects of alternariol: a mycotoxin.

Alternaria species are mycotoxin-producing fungi known to infect fresh produce and to cause their spoilage. Humans get exposed to fungal secondary metabolites known as mycotoxin via the ingestion of contaminated food. Alternariol (AOH) (C14H10O5) is an isocoumarins produced by different species of Alternaria including Alternaria alternata. AOH is often found in grain, fruits and fruits-based food products with high levels in legumes, nuts, and tomatoes. AOH was first discovered in 1953, and it is nowadays linked to esophagus cancer and endocrine disruption due to its similarity to estrogen. Although considered as an emerging mycotoxin with no regulated levels in food, AOH occurs in highly consumed dietary products and has been detected in various masked forms, which adds to its occurrence. Therefore, this comprehensive review was developed to give an overview on recent literature in the field of AOH. The current study summarizes published data on occurrence levels of AOH in different food products in the last ten years and evaluates those levels in comparison to recommended levels by the regulating entities. Such surveillance facilitates the work of health risk assessors and highlights commodities that are most in need of AOH levels regulation. In addition, the effects of AOH on cells and animal models were summarized in two tables; data include the last two-year literature studies. The review addresses also the main characteristics of AOH and the possible human exposure routes, the populations at risk, and the effect of anthropogenic activities on the widespread of the mycotoxin. The commonly used detection and control methods described in the latest literature are also discussed to guide future researchers to focus on mitigating mycotoxins contamination in the food industry. This review aims mainly to serve as a guideline on AOH for mycotoxin regulation developers and health risk assessors.

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Investigation of the mechanisms behind ochratoxin A-induced cytotoxicity in human astrocytes and the protective effects of N-acetylcysteine.

Ochratoxin A (OTA) is a type of mycotoxin commonly found in raw and processed foods. It is essential to be aware of this toxin, as it can harm your health if consumed in high quantities. OTA can induce toxic effects in various cell models. However, a more comprehensive understanding of the harmful effects of OTA on human astrocytes is required. This study evaluated OTA's neurotoxic effects on the Gibco® Human Astrocyte (GHA) cell line, its underlying mechanisms, and the antioxidant N-acetylcysteine (NAC) ability to prevent them. OTA exposure within 5-30 µM has induced concentration-dependent cytotoxicity. In the OTA-treated cells, the levels of reactive oxygen species (ROS) were found to be significantly increased, while the glutathione (GSH) contents were found to decrease considerably. The western blotting of OTA-treated cells has revealed increased Bax, cleaved caspase-9/caspase-3 protein levels, and increased Bax/Bcl-2 ratio. In addition, exposure to OTA has resulted in the induction of antioxidant responses associated with the protein expressions of Nrf2, HO-1, and NQO1. On the other hand, the pretreatment with NAC has partially alleviated the significant toxic effects of OTA. In conclusion, our findings suggest that oxidative stress and apoptosis are involved in the OTA-induced cytotoxicity in GHA cells. NAC could act as a protective agent against OTA-induced oxidative damage.

Rapid and simultaneous detection of five mycotoxins and their analogs with a gold nanoparticle-based multiplex immuno-strip sensor.

Mycotoxins, as secondary metabolites produced by fungi, have been the focus of researchers in various countries and are considered to be one of the major risk factors in agricultural products. There is an urgent need for a rapid, simple and high-performance method to detect residues of harmful mycotoxins in agricultural foods. We have developed a gold nanoparticle-based multiplexed immunochromatographic strip biosensor that can simultaneously detect fifteen mycotoxins in cereal samples. With this optimized procedure, five representative mycotoxins, deoxynivalenol (DON), zearalenone (ZEN), T-2 toxin (T-2), tenuazonic acid (TEA) and alternariol (AOH) were detected in the range of 0.91-4.77, 0.04-0.56, 0.11-0.68, 0.12-1.02 and 0.09-0.75 ng/mL, respectively. The accuracy and stability of these measurements were demonstrated by analysis of spiked samples with recoveries of 91.8%-115.3% and coefficients of variation <8.7%. In addition, commercially available samples of real cereals were tested using the strips and showed good agreement with the results verified by LC-MS/MS. Therefore, Our assembled ICA strips can be used for the simultaneous detection of 5 mycotoxins and their analogs (15 mycotoxins in total) in grain samples, and the results were consistent between different types of cereal foods, this multiplexed immunochromatographic strip biosensor can be used as an effective tool for the primary screening of mycotoxin residues in agricultural products.

Mycotoxin detection in selected medicinal plants using chromatographic techniques.

Mycotoxins are toxic mycological products that when consumed, absorbed or inhaled cause sickness or even the death of humans. Therefore, the present study aimed to evaluate the contamination levels of mycotoxins (aflatoxins, AFB1 , AFB2 , AFG1 , AFG2 , and ochratoxin A, OTA) in selected medicinal herbs and shrubs using thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). A total of 15 samples of medicinal herbs and shrubs were selected. Among them, four samples were aflatoxin contaminated while two samples were ochratoxin A contaminated. The highest level of aflatoxin was detected in Justicia adhathoda (4,704.94 ppb) through HPLC (153.4 ppb) and through TLC, while the lowest level of aflatoxin was detected in Pegnum harmala (205.1 ppb) through HPLC. Similarly, the highest level of OTA was detected in Dodonia viscosa (0.53 ppb) through HPLC (0.5 ppb) and through TLC, while the lowest level was detected in J. adhathoda (O.11 ppb) through HPLC (0.4 ppb) and through TLC. The OTA concentration was very low, being negligible and below permissible limits. The present study concludes that there is a potential risk for the consumption of herbal decoctions. Therefore, regular monitoring and proper management of mycotoxins, including aflatoxins and OTA, in herbal medicines are needed to ensure the safety of herbal drugs to protect consumers.

Assessment of fungal species in some spices using molecular study with detection of their mycotoxins using chromatography analysis.

Spices are food flavouring agents that are highly used in Iraq. However, they may be contaminated by toxicogenic fungi and subsequent production of mycotoxins. The aim of this study was to investigate the contamination of commonly used spices with fungi using polymerase chain reaction (PCR) assay and to detect fungal mycotoxin using high-performance liquid chromatography. Thirty-five spices (seven samples from each black pepper, red pepper, turmeric, cumin and ginger) were cultured on an appropriate medium to identify various fungi species. Later on, the toxigenicity of Aspergillus flavus and Aspergillus niger was determined using a PCR assay. The fungal mycotoxins, including aflatoxins and ochratoxins, were then determined through a high-performance liquid chromatography using the validated Quick, Easy, Cheap, Effective, Rugged, and Safe (QuECHERS) method. Aspergillus species were the predominantly isolated fungi, followed by Penicillium and Fusarium. The PCR results indicate the high toxigenicity of A. flavus as 85.7% of the strains had aflQ/aflR genes and 79% had PKS15KS/PKS15C-MeT genes. Regarding mycotoxin contamination in spices, the highest rates of aflatoxins and ochratoxins were found in black pepper (5.913 µg/kg) and red chilli (6.9055 µg/kg), respectively. Spices are susceptible substrates for the growth of mycotoxigenic fungi. Thus, regular effective surveillance and quality control procedures are highly recommended.

Anti-mycotoxin feed additives: effects on metabolism, mycotoxin excretion, performance, and total tract digestibility of dairy cows fed artificially multi-mycotoxin-contaminated diets.

The aim of this study was to evaluate the effects of different anti-mycotoxin feed additives on the concentration of mycotoxins in milk, urine, and blood plasma of dairy cows fed artificially multi-mycotoxin-contaminated diets. Secondarily, performance, total-tract apparent digestibility of nutrients, and blood parameters were evaluated. Twelve multiparous cows (165 ± 45 d in milk, 557 ± 49 kg body weight, and 32.1 ± 4.57 kg/d milk yield at the start of the experiment) were blocked according to parity, milk yield, and days in milk and used in a 4 × 4 Latin square design experiment with 21-d periods, where the last 7 d were used for sampling and data analysis. Treatments were: 1) Mycotoxin group (MTX), basal diet (BD) without anti-mycotoxin feed additives; 2) Hydrated sodium calcium aluminosilicate (HSCA), HSCA added to the BD at 25g/cow/d; 3) Mycotoxin deactivator 15 (MD15), MD (Mycofix® Plus, dsm-firmenich) added to the BD at 15 g/cow/d; and 4) Mycotoxin deactivator 30 (MD30), MD added to the BD at 30 g/cow/d. Cows from all treatments were challenged with a blend of mycotoxins containing 404 µg aflatoxins B1 (AFB1), 5,025 µg deoxynivalenol (DON), 8,046 µg fumonisins (FUM), 195 µg T2 toxin (T2), and 2,034 µg of zearalenone (ZEN) added daily to the BD during the last 7 d of each period. Neither performance (milk yield and composition) nor nutrient digestibility was affected by treatments. All additives reduced aflatoxin M1 (AFM1) concentration in milk, whereas MD15 and MD30 group had lower excretion of AFM1 in milk than HSCA. DON, FUM, T2, or ZEN were not detected in milk of MD15 and MD30. Concentrations in milk of DON, FUM, T2, and ZEN were similar between MTX and HSCA. Except for AFM1, none of the analyzed mycotoxins were detected in urine of MD30 group. Comparing HSCA to MD treatments, the concentration of AFM1 was greater for HSCA, whereas MD30 was more efficient at reducing AFM1 in urine than MD15. AFM1, DON, FUM, and ZEN were not detected in the plasma of cows fed MD30, and DON was also not detected in MD15 group. Plasma concentration of FUM was lower for MD15, similar plasma FUM concentration was reported for HSCA and MTX. Plasma concentration of ZEN was lower for MD15 than MTX and HSCA. Serum concentrations of haptoglobin and hepatic enzymes were not affected by treatments. Blood concentration of sodium was lower in HSCA compared with MD15 and MD30 groups. In conclusion, the mycotoxin deactivator proved to be effective in reducing the secretion of mycotoxins in milk, urine, and blood plasma, regardless of the dosage. This reduction was achieved without adverse effects on milk production or total-tract digestibility in cows fed multi-mycotoxin-contaminated diets over a short-term period. Greater reductions in mycotoxin secretion were observed with full dose of MD.

Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

SeMet alleviates AFB1-induced oxidative stress and apoptosis in rabbit kidney by regulating Nrf2//Keap1/NQO1 and PI3K/AKT signaling pathways.

The purpose of this study was to explore the protective effect of SeMet on renal injury induced by AFB1 in rabbits and its molecular mechanism. Forty rabbits of 35 days old were randomly divided into control group, AFB1 group (0.3 mg AFB1/kg b.w), 0.2 mg/kg Se + AFB1 group (0.3 mg AFB1/kg b.w + 0.2 mg SeMet/kg feed) and 0.4 mg/kg Se + AFB1 group (0.3 mg AFB1/kg b.w + 0.4 mg SeMet/kg feed). The SeMet treatment group was fed different doses of SeMet diets every day for 21 days. On the 17-21 day, the AFB1 treatment group, the 0.2 mg/kg Se + AFB1 group and the 0.4 mg/kg Se + AFB1 group were administered 0.3 mg AFB1 /kg b.w by gavage (dissolved in 0.5 ml olive oil) respectively. The results showed that AFB1 poisoning resulted in the changes of renal structure, the increase of renal coefficient and serum biochemical indexes, the ascent of ROS and MDA levels, the descent of antioxidant enzyme activity, and the significant down-regulation of Nrf2, HO-1 and NQO1. Besides, AFB1 poisoning increased the number of renal apoptotic cells, rised the levels of PTEN, Bax, Caspase-3 and Caspase-9, and decreased the levels of PI3K, AKT, p-AKT and Bcl-2. In summary, SeMet was added to alleviate the oxidative stress injury and apoptosis of kidney induced by AFB1, and the effect of 0.2 mg/kg Se + AFB1 is better than 0.4 mg/kg Se + AFB1.