Development of a reusable polymeric fluorescence sensor based on acryloyl ß-cyclodextrin for the determination of aflatoxin B1 in grain products.

AFB1 is a harmful substance that can be found in agricultural products and can seriously affect human health, even in trace amounts. Therefore, monitoring AFB1 levels to ensure food safety and protect public health is crucial. New, highly reliable, selective, and rapid detection methods are needed to achieve this goal. Our work involves the development of a polymeric membrane sensor using radical polymerization that can accurately detect AFB1. Various spectroscopic techniques (Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM)) were used to obtain information about the structural and morphological properties of the prepared sensor. The sensor displayed fluorescence selectively responsive to AFB1 at the excitation wavelength of 376 nm and emission wavelength of 423 nm. The polymeric fluorescence sensor showed good sensitivity and a wide linear range from 9.61 × 10-10 and 9.61 × 10-9 mol/L for AFB1quantification. The limit of detection (LOD) is as low as 3.84 × 10-10 mol/L for AFB1. Other mycotoxins, such as aflatoxin B2 and aflatoxin G1, did not interfere with the sensor's high selectivity towards AFB1. To test the sensor's effectiveness in detecting AFB1 in real samples, three different grain samples - peanuts, hazelnut butter, and peanuts with a sauce known to contain AFB1 - were utilized. The results were satisfactory and demonstrated that the sensor can be successfully employed in real samples, with an error range of 0.43 % to 12.10 %.

Integration of Fe-MOF-laccase-magnetic biochar: From Rational Designing of a biocatalyst to aflatoxin B1 decontamination of peanut oil.

Enzymatic degradation of aflatoxins in food commodities has gained significant attention. However, enzyme denaturation in organic media discourages their direct use in oils to remove aflatoxins. For that, enzymes are immobilized or encapsulated for improved stability and reusability under unfavorable conditions. We sandwiched the laccase between a carrier and an outer protective layer. We used spent-mushroom-substrate (SMS) derived porous magnetic biochar as the laccase carrier and coated it with an iron MOF to create a biocomposite, Fe-BTC@Lac@FB. The immobilized laccase demonstrated enhanced chemical, thermal, and storage stability and proficient reusability. Fe-BTC@Lac@FB exhibited 11 times enhanced aflatoxin B1 (AFB1) degradation compared to free laccase (FL). In addition, thermally inactivated Fe-BTC@Lac@FB could adsorb 11.2 mg/g of AFB1 from peanut oil. Multi-aflatoxin removal also proved promising, while Fe-BTC@Lac@FB could retain > 85 % of AFB1 removal efficacy after five reusability cycles. Fe-BTC@Lac@FB treatment did not affect peanut oil quality as indicated by different oil quality parameters and proved essentially non-cytotoxic. All these aspects helped recognize Fe-BTC@Lac@FB as an excellent laccase-carrying material with exceptionally higher stability, activity, and reusability.

Signal-enhanced electrochemical sensor employing MWCNTs/CMK-3/AuNPs and Au@Pd core-shell structure for sensitive determination of AFB1 in complex matrix.

A sandwich electrochemical sensor was fabricated based on multi-walled carbon nanotubes/ordered mesoporous carbon/AuNP (MWCNTs/CMK-3/AuNP) nanocomposites and porous core-shell nanoparticles Au@PdNPs to achieve rapid and sensitive detection of AFB1 in complex matrices. MWCNTs/CMK-3/AuNP nanocomposite, which was prepared by self-assembly method, served as a substrate material to increase the aptamer loading and improve the conductivity and electrocatalytic activity of the electrode for the first signal amplification. Then, Au@PdNPs, which were synthesized by one-pot aqueous phase method, were applied as nanocarriers loaded with plenty of capture probe antibody (Ab) and signal molecule toluidine blue (Tb) to form the Au@PdNPs-Ab-Tb bioconjugates for secondary signal amplification. The sensing system could still significantly improve the signal output intensity even in the presence of ultra-low concentration target compound due to the dual signal amplification of MWCNTs/CMK-3/AuNP nanocomposites and Au@PdNPs-Ab-Tb. The method exhibited high selectivity, low detection limit (9.13 fg/mL), and strong stability to differentiate AFB1 from other mycotoxins. Furthermore, the sensor has been successfully applied to the quantitative determination of AFB1 in corn, malt, and six herbs, which has potential applications in food safety, quality control, and environmental monitoring.

Elastin-like polypeptide-functionalized nanobody for column-free immunoaffinity purification of aflatoxin B1.

A column-free immunoaffinity purification (CFIP) technique for sample preparation of aflatoxin B1 (AFB1) was developed using an AFB1-specific nanobody (named G8) and an elastin-like polypeptide (ELP). The reversible phase transition between liquid and solid in response to temperature changes was exhibited by the ELP which was derived from human elastin. The G8 was tagged with ELPs of various lengths (20, 40, 60, and 80 repeat units) at the C-terminus using recursive directional ligation (RDL). Coding sequences were then subcloned into pET30a at the multiple cloning sites. Bioactive recombinant proteins were produced by expressing them as inclusion bodies in Escherichia coli BL21 (DE3), then dissolved and refolded. Analysis by indirect competitive enzyme-linked immunosorbent assay (icELISA) and transition temperature (Tt) measurement confirmed that the refolded G8-ELPs preserved the ability to recognize AFB1 as well as phase transition when the temperature rose above Tt. To establish the optimal conditions for cleaning AFB1, the effects of various parameters on recovery were investigated. The recovery in ELISA tests was 95 ± 3.67% under the optimized CFIP workflow. Furthermore, the CFIP-prepared samples were applied for high-performance liquid chromatography (HPLC) detection. The recovery in the CFIP-HPLC test ranged from 54 ± 1.86% to 98 ± 3.58% for maize, rice, soy sauce, and vegetable oil samples. To the best of our knowledge, this is the first report combining the function of both nanobody and ELP to develop a cleanup technique for small molecules in a complex matrix. The CFIP for the sample pretreatment was easy to use and inexpensive. In contrast to conventional immunosensitivity materials, the reagent utilized in the CFIP was entirely biosynthesized without any chemical coupling reactions. This suggests that the nanobody-ELP may serve as a useful dual-functional reagent for the development of sample cleaning or purification methods.

Aflatoxin exposure is associated with an increased risk of gallbladder cancer.

Gall bladder cancer (GBC) is common among the socioeconomically deprived populations of certain geographical regions. Aflatoxin is a genotoxic hepatocarcinogen, which is recognized to have a role in the pathogenesis of hepatocellular carcinoma. However, the role of aflatoxin in the pathogenesis of GBC is largely unknown. We determined serum AFB1-Lys albumin adduct (AAA) levels as a marker of aflatoxin exposure in the patients with GBC and compared to those without GBC. The relationship of AAA levels to cytogenetic (TP53mutation&HER2/neu amplification) and radiological characteristics of the tumor was assessed. We included GBC cases (n = 51) and non-GBC controls (n = 100). Mean serum AAA levels were higher in the GBC group (n = 51) than those without GBC (n = 100) (26.1 ± 12.2 vs. 13.1 ± 11.9 ng/mL; p < .001). HER2/neu expression was associated with higher AAA levels compared to those with equivocal or negative expression (43.9 ± 3 vs. 28.6 ± 10 vs. 19.3 ± 7 ng/mL; p < .001). Older age (age >50 years) (odds ratio [OR] = 3.2 [CI: 1.3-8.2]; p = .013), positive Helicobacter pylori serology (OR = 5.1 [CI: 1.4-17.8]; p = .012), presence of GS (OR = 5 [CI: 1.5-16.9]; p = .009) and detectable AAA levels (OR = 6.8 [CI: 1.3-35.7]; p = .024) were independent risk factors for the presence of the GBC among all study subjects. Among patients harboring GS, older age (age >50 years) (OR = 4.5 [CI: 1.3-14.9]; p = .015), female gender (OR = 3.8 [CI: 1.2-12.5]; p = .027), presence of multiple GS (OR = 21.9 [CI: 4.8-100.4]; p < .001) and high serum AAA levels (OR = 5.3 [CI: 1.6-17.3]; p = .006) were independent risk factors for the presence of the GBC. Elderly age >50 years (OR = 2.6 [CI: 1.3-5.2]; p = .010) and frequent peanut consumption (OR = 2.3 [CI: 1.1-4.9]; p = .030) were independent risk factors for high serum AAA levels. The current study has implications for the prevention of GBC through the reduction of dietary aflatoxin exposure.

Mitigating CYP3A4-mediated aflatoxin toxicity with algal-derived Sodium Copper Chlorophyllin: Production and In-silico insights.

The present research explores the cytotoxic mechanism of protein Cytochrome P450 (CYP3A4) with aflatoxin (AFB1), a potent carcinogen. Cytochrome P450 is an essential enzyme involved in drug metabolism, however epoxide formation due to the binding event of AFB1 leads to cell cytotoxicity. In this direction, our study elucidates the scavenging effect of algal-derived Sodium Copper Chlorophyllin (SCC) over AFB1 cytotoxicity. Cyanobacteria/ microalgae-derived SCC have garnered attention due to its diverse applications in pharmacological and food industries. This work began with the production of SCC from Spirulina and Chlorella sp. over a stipulated period of growth. Subsequently, the study delved into the interplay between SCC and the carcinogenic impact of AFB1 on the CYP3A4 enzyme. Computational studies demonstrated SCC binding and blocking mechanisms against AFB1. Our research intended to determine whether CYP3A4 can bind to SCC that, in turn, act as an interceptor for AFB1 or influence the metabolism of bound AFB1. Current results support that SCC is an effective AFB1 trap as it shows interactions with AFB1. These findings would open up new avenues in clinical biology/pharmacology to further explore the mechanisms of action of CYP3A4 with AFB1 and SCC, offering promising prospects for abating cell cytotoxicity.

Effects of alfa lipoic acid and coenzyme Q10 treatment on AFB1-induced oxidative, inflammatory, and DNA damages in rats.

Food contamination with Aflatoxin B1 (AFB1) is a worldwide concern that adversely affects animal and human health. The study aimed to evaluate the protective effect of alpha lipoic acid (ALA) and/or co-enzyme Q10 (CQ10) against the harmful effects of AFB1 on the liver and kidneys. Fifty-six mature male Wistar Albino rats (180-200 g) were divided into seven groups, each with eight rats: (1) saline was given as a control, (2) ALA (100 mg/kg bw/day) was given by stomach gavage for fifteen days, and (3) CQ10 (10 mg/kg bw/day) was given by stomach gavage for fifteen days. Group (4) orally given AFB1 (2.5 mg/kg bw) on days 12th and 14th, (5) received AFB1 and ALA, (6) received AFB1 and CQ10, and (7) received AFB1, ALA, and CQ10, as previously described in the ALA, CQ10, and AFB1 groups. After the exposure to AFB1, a significant increase in liver markers (AST, ALT, ALP, and LDH) and renal function tests (BUN and creatinine) was observed compared with the control. ALA and/or CQ10 significantly reduced enzymes of liver and renal functions, as compared with AFB1. AFB1 exposure threw off the balance between oxidants and antioxidants. Still, ALA and/or CQ10 made oxidative stress (MDA, NO, and 8-OHdG) much lower and antioxidant activities (GSH, GSH-Px, SOD, and CAT) much higher. When we used the two together, the activities matched the control levels. Interestingly, this study shows that ALA and CQ10 significantly lowered IL-1ß, IL-6, and TNF-α levels compared to the control values when used together after AFB1 exposure caused robust inflammation. Some CQ10 treatment parameters significantly outperformed those of ALA. ALA and CQ10 together worked better than either one alone to protect against AFB1-induced toxicity in the hepatic and renal parenchyma in terms of reducing inflammation, preventing DNA damage, and fighting free radicals.

Widespread distribution of the DyP-carrying bacteria involved in the aflatoxin B1 biotransformation in Proteobacteria and Actinobacteria.

Aflatoxin is one of the most notorious mycotoxins, of which aflatoxin B1 (AFB1) is the most harmful and prevalent. Microbes play a crucial role in the environment for the biotransformation of AFB1. In this study, a bacterial consortium, HS-1, capable of degrading and detoxifying AFB1 was obtained. Here, we combined multi-omics and cultivation-based techniques to elucidate AFB1 biotransformation by consortium HS-1. Co-occurrence network analysis revealed that the key taxa responsible for AFB1 biotransformation in consortium HS-1 mainly belonged to the phyla Proteobacteria and Actinobacteria. Moreover, metagenomic analysis showed that diverse microorganisms, mainly belonging to the phyla Proteobacteria and Actinobacteria, carry key functional enzymes involved in the initial step of AFB1 biotransformation. Metatranscriptomic analysis indicated that Paracoccus-related bacteria were the most active in consortium HS-1. A novel bacterium, Paracoccus sp. strain XF-30, isolated from consortium HS-1, contains a novel dye-decolorization peroxidase (DyP) enzyme capable of effectively degrading AFB1. Taxonomic profiling by bioinformatics revealed that DyP, which is involved in the initial biotransformation of AFB1, is widely distributed in metagenomes from various environments, primarily taxonomically affiliated with Proteobacteria and Actinobacteria. The in-depth examination of AFB1 biotransformation in consortium HS-1 will help us to explore these crucial bioresources more sensibly and efficiently.

Aflatoxin B1-associated oxidative stress along with toxicopathological and immunological alterations is efficiently counteracted by dietary supplementation of distillery yeast sludge in broilers.

Aflatoxin B1 (AFB1) is among the most potent genotoxic and carcinogenic mycotoxins and is a major source of distress for the growing poultry sector. On the other hand, distillery yeast sludge or distillery sludge (DS) is a byproduct of molasses-based industries. It is often treated as a waste despite containing abundant nutrients particularly protein, basic amino acids, and vitamins along with other macro and micronutrients. This study was designed to investigate the oxidative stress and immunological alterations induced by AFB1 and their amelioration by dietary supplementation with DS. For this purpose, 360 newly hatched broiler chicks were randomly divided into twelve groups (30 birds each) and fed different combinations of AFB1 (100, 200, or 600 µg/kg) and DS (5 or 10 g/kg) for 42 days. The parameters under consideration were body weight, feed conversion ratio (FCR), relative organ weights, histopathological examination of different visceral organs, total antioxidant capacity, antibody response to intravenous injection of sheep red blood cells, in situ lymphoproliferative response to phytohemagglutinin-P, and phagocytic potential through a carbon clearance assay system. The results of this study established that DS supplementation ameliorated AFB1-associated oxidative stress and ameliorated toxicopathological and immunological anomalies in groups given AFB1 at 100 µg/kg and 200 µg/kg; however, little to no relief was observed in birds fed AFB1 at 600 µg/kg. The determination of the actual ratio of the AFB1 to the DS for substantiating the ameliorating effects requires further investigation.

Rhein Inhibits Cell Development and Aflatoxin Biosynthesis via Energy Supply Disruption and ROS Accumulation in Aspergillus flavus.

Aspergillus flavus and its carcinogenic secondary metabolites, aflatoxins, not only cause serious losses in the agricultural economy, but also endanger human health. Rhein, a compound extracted from the Chinese herbal medicine Rheum palmatum L. (Dahuang), exhibits good anti-inflammatory, anti-tumor, and anti-oxidative effects. However, its effect and underlying mechanisms against Aspergillus flavus have not yet been fully illustrated. In this study, we characterized the inhibition effect of rhein on A. flavus mycelial growth, sporulation, and aflatoxin B1 (AFB1) biosynthesis and the potential mechanism using RNA-seq analysis. The results indicate that A. flavus mycelial growth and AFB1 biosynthesis were significantly inhibited by 50 µM rhein, with a 43.83% reduction in colony diameter and 87.2% reduction in AFB1 production. The RNA-seq findings demonstrated that the differentially expressed genes primarily participated in processes such as spore formation and development, the maintenance of cell wall and membrane integrity, management of oxidative stress, the regulation of the citric acid cycle, and the biosynthesis of aflatoxin. Biochemical verification experiments further confirmed that 50 µM rhein effectively disrupted cell wall and membrane integrity and caused mitochondrial dysfunction through disrupting energy metabolism pathways, leading to decreased ATP synthesis and ROS accumulation, resulting in impaired aflatoxin biosynthesis. In addition, a pathogenicity test showed that 50 µM rhein inhibited A. flavus spore growth in peanut and maize seeds by 34.1% and 90.4%, while AFB1 biosynthesis was inhibited by 60.52% and 99.43%, respectively. In conclusion, this research expands the knowledge regarding the antifungal activity of rhein and provides a new strategy to mitigate A. flavus contamination.

Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives.

Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.

Characterization, Mechanism, and Application of Dipeptidyl Peptidase III: An Aflatoxin B1-Degrading Enzyme from Aspergillus terreus HNGD-TM15.

Aflatoxin B1 is a notorious mycotoxin with mutagenicity and carcinogenicity, posing a serious hazard to human and animal health. In this study, an AFB1-degrading dipeptidyl-peptidase III mining from Aspergillus terreus HNGD-TM15 (ADPP III) with a molecular weight of 79 kDa was identified. ADPP III exhibited optimal activity toward AFB1 at 40 °C and pH 7.0, maintaining over 80% relative activity at 80 °C. The key amino acid residues that affected enzyme activity were identified as H450, E451, H455, and E509 via bioinformatic analysis and site-directed mutagenesis. The degradation product of ADPP III toward AFB1 was verified to be AFD1. The zebrafish hepatotoxicity assay verified the toxicity of the AFB1 degradation product was significantly weaker than that of AFB1. The result of this study proved that ADPP III presented a promising prospect for industrial application in food and feed detoxification.

Antioxidant, anti-inflammatory, and anti-DNA damage effects of carnosic acid against aflatoxin B1-induced hepatic, renal, and cardiac toxicities in rats.

Background: Aflatoxin B1 (AFB1) food contamination is a global health hazard that has detrimental effects on both human and animal health. The objective of the current study is to assess the protective impact of carnosic acid against AFB1-induced toxicities in the liver, kidneys, and heart. Methods: Forty male Wistar Albino rats (weighting 180 ~ 200 g) were allocated into 5 groups (8 rats each); the 1st group received saline as served as a control, the 2nd group received carnosic acid (CA100) at a dose of 100 mg/kg bw/day by gavage for 14 days, the 3rd group received AFB1 at a dose of 2.5 mg/kg bw, orally twice on days 12 and 14, the 4th group (AFB1-CA50) received AFB1 as in the 3rd group and CA at a dose of 50 mg/kg bw/day, and the 5th group (AFB1-CA100) received AFB1 as in the 3rd group and CA as in the 2nd group. Results: CA significantly decreased the liver enzymes (ALT, AST. ALP), renal function products (LDH, BUN, creatinine), and cardiac enzymes (CK and CK-MB) to control levels after the high increment by AFB1 exposure. Moreover, CA significantly decreased the oxidative stress (MDA, NO, 8-OHdG) and increased the antioxidant enzyme activities (CAT, GSH, GSH-Px, and SOD) after severe disruption of oxidant/antioxidant balance by AFB1 exposure. Interestingly, CA significantly decreased the proinflammatory mediators (IL-6, IL-1ß, and TNF-α) to the control levels after severe inflammation induced by AFB1 exposure. Conclusions: Conclusively, CA had antioxidant, anti-inflammatory, and anti-DNA damage effects against hepatic, renal, and cardiac AFB1-induced toxicities.

One stone two birds: Recycling of an agri-waste to synthesize laccase-immobilized hierarchically porous magnetic biochar for efficient degradation of aflatoxin B1 in aqueous solutions and corn oil.

Aflatoxin B1 (AFB1) contamination of oils is a serious concern for the safety of edible oil consumers. Enzyme-assisted detoxification of AFB1 is an efficient and safe method for decontaminating oils, but pristine enzymes are unstable in oils and require modifications before use. Therefore, we designed a novel and magnetically separable laccase-carrying biocatalyst containing spent-mushroom-substrate (SMS)-derived biochar (BF). Laccase was immobilized on NH2-activated magnetic biochar (BF-NH2) through covalent crosslinking, which provided physicochemical stability to the immobilized enzyme. After 30 days of storage at 4 °C, the immobilized laccase (product named "BF-NH2-Lac") retained ~95 % of its initial activity, while after five repeated cycles of ABTS oxidation, ~85 % activity retention was observed. BF-NH2-Lac was investigated for the oxidative degradation of AFB1, which exhibited superior performance compared to free laccase. Among many tested natural compounds as mediators, p-coumaric acid proved the most efficient in activating laccase for AFB1 degradation. BF-NH2-Lac demonstrated >90 % removal of AFB1 within 5.0 h, while the observed degradation efficiency in corn oil and buffer was comparable. An insight into the adsorptive and degradative removal of AFB1 revealed that AFB1 removal was governed mainly by degradation. The coexistence of multi-mycotoxins did not significantly affect the AFB1 degradation capability of BF-NH2-Lac. Investigation of the degradation products revealed the transformation of AFB1 into non-toxic AFQ1, while corn oil quality remained unaffected after BF-NH2-Lac treatment. Hence, this study holds practical importance for the research, knowledge-base and industrial application of newly proposed immobilized enzyme products.

In silico studies and development of a protein-based electrochemical sensor for selective and sensitive detection of aflatoxin B1.

Proteins from different species have been docked with aflatoxin B1 (AFB1) and identified 3 proteins (prostaglandin-E(2)9-reductase from Oryctolagus uniculus, proto-oncogene serine/threonine-protein kinase Pim-1 and human immunoglobulin G (hIgG)) as potential candidates to develop an electrochemical sensor. Fluorescence spectroscopy experiments have confirmed the interaction of hIgG with AFB1 with an affinity constant of 4.6 × 105 M-1. As a proof-of-concept, hIgG was immobilized on carbon nanocomposite (carbon nanotube-nanofiber, CNT-F)-coated glassy carbon electrode (GCE). FT-IR spectra, HR-TEM and BCA assay have confirmed successful immobilization of hIgG on the electrode (hIgG@CNT-F/GCE). The preparation of this protein electrochemical sensor requires only 1 h 36 min, which is fast as compared with preparing an electro immunosensor. hIgG@CNT-F/GCE has displayed an excellent AFB1 limit of detection (0.1 ng/mL), commendable selectivity in the presence of two other mycotoxins (ochratoxin A and patulin) and the detection of  AFB1 in spiked peanuts and corn samples.

Self-designed portable dual-mode fluorescence device with custom python-based analysis software for rapid detection via dual-color FRET aptasensor with IoT capabilities.

An innovative aptasensor incorporating MoS2-modified bicolor quantum dots and a portable spectrometer, designed for the simultaneous detection of ochratoxin A (OTA) and aflatoxin B1 (AFB1) in corn was developed. Carbon dots and CdZnTe quantum dots were as nano-donors to label OTA and AFB1 aptamers, respectively. These labeled aptamers were subsequently attached to MoS2 receptors, enabling fluorescence resonance energy transfer (FRET). With targets, the labeled aptamers detached from the nano-donors, thereby disrupting the FRET process and resulting in fluorescence recovery. Furthermore, a portable dual-mode fluorescence detection system, complemented with customized python-based analysis software, was developed to facilitate rapid and convenient detection using this dual-color FRET aptasensor. The developed host program is connected to the spectrometer and transmits data to the cloud, enabling the device to have Internet of Things (IoT) characteristics. Connected to the cloud, this IoT-enabled device offers convenient and reliable fungal toxin detection for food safety.

The Effects of Aflatoxin B1 on Liver Cholestasis and Its Nutritional Regulation in Ducks.

The aim of this study was to investigate the effects of aflatoxin B1 (AFB1) on cholestasis in duck liver and its nutritional regulation. Three hundred sixty 1-day-old ducks were randomly divided into six groups and fed for 4 weeks. The control group was fed a basic diet, while the experimental group diet contained 90 µg/kg of AFB1. Cholestyramine, atorvastatin calcium, taurine, and emodin were added to the diets of four experimental groups. The results show that in the AFB1 group, the growth properties, total bile acid (TBA) serum levels and total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px), and glutathione (GSH) liver levels decreased, while the malondialdehyde (MDA) and TBA liver levels increased (p < 0.05). Moreover, AFB1 caused cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin could reduce the TBA serum and liver levels (p < 0.05), alleviating the symptoms of cholestasis. The qPCR results show that AFB1 upregulated cytochrome P450 family 7 subfamily A member 1 (CYP7A1) and cytochrome P450 family 8 subfamily B member 1 (CYP8B1) gene expression and downregulated ATP binding cassette subfamily B member 11 (BSEP) gene expression in the liver, and taurine and emodin downregulated CYP7A1 and CYP8B1 gene expression (p < 0.05). In summary, AFB1 negatively affects health and alters the expression of genes related to liver bile acid metabolism, leading to cholestasis. Cholestyramine, atorvastatin calcium, taurine, and emodin can alleviate AFB1-induced cholestasis.

Exploring the Efficacy of Using Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus, Clay Minerals, and Walnut Nutshells for Mycotoxin Remediation.

The aim of this study was to evaluate the effectiveness of nine different biological compounds to reduce mycotoxins concentrations. The hypothesis of this study was that a static in vitro gastrointestinal tract model, as an initial screening tool, can be used to simulate the efficacy of Geotrichum fermentans, Rhodotorula rubra, Kluyveromyce marxiamus yeast cell walls and their polysaccharides, red and white clay minerals, and walnuts nutshells claiming to detoxify AFB1, ZEA, DON, and T-2 toxin mycotoxins. Mycotoxin concentrations were analyzed using high-performance liquid chromatography (HPLC) with fluorescent (FLD) and ultraviolet detectors (UV). The greatest effects on reducing mycotoxin concentrations were determined as follows: for AFB1, inserted G. fermentans cell wall polysaccharides and walnut nutshells; for ZEA, inserted R. rubra and G. fermentans cell walls and red clay minerals; for DON, R. rubra cell wall polysaccharides and red clay minerals; and for T-2 toxin, R. rubra cell walls, K. marxianus, and G. fermentans cell wall polysaccharides and walnut nutshells. The present study indicated that selected mycotoxin-detoxifying biological compounds can be used to decrease mycotoxin concentrations.

A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

The Autophagy-Related Protein ATG8 Orchestrates Asexual Development and AFB1 Biosynthesis in Aspergillus flavus.

Autophagy, a conserved cellular recycling process, plays a crucial role in maintaining homeostasis under stress conditions. It also regulates the development and virulence of numerous filamentous fungi. In this study, we investigated the specific function of ATG8, a reliable autophagic marker, in the opportunistic pathogen Aspergillus flavus. To investigate the role of atg8 in A. flavus, the deletion and complemented mutants of atg8 were generated according to the homologous recombination principle. Deletion of atg8 showed a significant decrease in conidiation, spore germination, and sclerotia formation compared to the WT and atg8C strains. Additionally, aflatoxin production was found severely impaired in the ∆atg8 mutant. The stress assays demonstrated that ATG8 was important for A. flavus response to oxidative stress. The fluorescence microscopy showed increased levels of reactive oxygen species in the ∆atg8 mutant cells, and the transcriptional result also indicated that genes related to the antioxidant system were significantly reduced in the ∆atg8 mutant. We further found that ATG8 participated in regulating the pathogenicity of A. flavus on crop seeds. These results revealed the biological role of ATG8 in A. flavus, which might provide a potential target for the control of A. flavus and AFB1 biosynthesis.