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The current study tries to find the impact of the integration of laccase enzyme (Lac) onto magnetized chitosan (Cs) nanoparticles composed of molybdenum disulfide (MoS2 NPs) (Fe3O4/Cs/MoS2/Lac NPs) on the removal of AFM1 in milk samples. The Fe3O4/Cs/MoS2/Lac NPs were characterized by FT-IR, XRD, BET, TEM, FESEM, EDS, PSA, and VSM analysis. The cytotoxic activity of the synthesized nanoparticles in different concentrations was evaluated using the MTT method. The results show that the synthesized nanoparticles don't have cytotoxic activity at concentrations less than 20 mg/l. The ability of the prepared nanoparticles to remove AFM1 was compared by bare laccase enzyme, MoS2, and Fe3O4/Cs/MoS2 composite, indicating that the Fe3O4/Cs/MoS2/Lac NPs the highest adsorption efficiency toward AFM1. Besides, the immobilization efficiency of laccase with a concentration range of 0.5-2.0 was investigated, indicating that the highest activity recovery of 96.8% was obtained using 2 mg/ml laccase loading capacity. The highest removal percentage of AFM1 (68.5%) in the milk samples was obtained by the Fe3O4/Cs/MoS2/Lac NPs at a contact time of 1 h. As a result, Fe3O4/MoS2/Cs/Lac NPs can potentially be utilized as an effective sorbent with high capacity and selectivity to remove AFM1 from milk samples.

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Milk is an essential part of the human diet and is a nutrient-rich food that improves nutrition and food security. The aim of this study was to determine the presence and concentration of aflatoxin M1 (AFM1), adulterants, microbial loads, and physicochemical properties of raw cow's milk (CM) in Nekemte City, Ethiopia. A total of 12 samples of fresh CM were purposefully collected from four kebeles in the city (Bake Jama, Burka Jato, Cheleleki, and Bakanisa Kese) based on the potential of each milk production and distributor site. The AFM1 concentration was determined by high-performance liquid chromatography (HPLC) with a Sigma-Aldrich standard (St. Louis, MO, USA). The concentrations of AFM1 in Bake Jama, Burka Jato, Cheleleki, and Bakanisa Kese were found to be 0.01-0.03 g/L, 0.31-0.35 g/L, 0.19-0.21 g/L, and 0.04-0.07 g/L, respectively. The concentrations of AFM1 in the present study varied significantly (p < 0.05) and ranged from 0.01 g/L to 0.35 g/L. These results show that of the 12 samples tested, all were positive for AFM1 and contaminated to varying degrees. The results of this study also revealed that the concentration of AFM1 in 7 (58%) of the 12 milk samples was above the European Union's (EU) maximum tolerance limit (0.05 g/L). The present study also revealed that of the investigated adulterants, only the addition of water had positive effects on three milk samples, while the remaining adulterants were not detected in any of the milk samples. The total bacterial count (TBC) and total coliform count (TCC) were significantly (p < 0.05) different and ranged from 5.53 to 6.82 log10cfumL-1 and from 4.21 to 4.74 log10cfumL-1, respectively. The physicochemical properties of the milk samples in the present study were significantly (p < 0.05) different and ranged from 2.8% to 5.75% fat, 7.03% to 9.75% solid-not-fat (SNF), 2.35% to 3.61% protein, 3.33% to 5.15% lactose, 11.54% to 13.69% total solid, 0.16% to 0.18% titratable acid, 26.7 to 32.1°C, 6.35 to 6.55 pH, and 1.027 to 1.030 specific gravity. The physicochemical parameters of the raw milk in the study area met the required quality standards. Hence, further studies are required to determine the extent of the problem and the factors associated with high levels of AFM1 in raw milk in the study areas, including the detection of aflatoxin B1 (AFB1) in animal feed.

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The numerous strategies have been conducted worldwide to mitigate the presence of these hazardous toxins. In this systematic study, these researches are summarized. The search of this study was done with keywords aflatoxin M1, AFM1, reduce, decrease, mitigation, prevent, prevention, and milk in databases without a time limit. A total of 49 manuscripts were carefully reviewed, and their data were extracted. Some interventions focused on modifying animal rations, aiming to reduce AFM1 in milk. Some were applied directly to the animals. In this method, which was done more than other research interventions, some toxin binders are used as feed additives. The third type of intervention consisted of measures that were taken directly on the milk itself. Among the three types of interventions, the use of toxin binders in animal feed was more practical and effective.

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Microorganisms are the only entities in the biosphere with an incomparable ability to employ diverse organic and inorganic compounds for growth and convert it to simple form that is no longer harmful to human health and environment. Food grade microorganisms such as lactic acid bacteria, bifidobacteria, propionibacteria as well as several yeast species are associated with food fermentation processes as well as have gained probiotic status owing to their noteworthy offerings in health stimulation as a natural gut microbiota in animals and humans. However, as biological agents little is known about their application for bioremediation and biotransformation aptitude. In context to this, aflatoxin M1 is a class of mycotoxins often associated with milk through consumption of fungus contaminated feed & fodders by cattle and well documented for their adverse health effects. Therefore, current review summarizes significance of aflatoxins present in milk and dairy products in human life, their source, types & health implications; food grade bacteria including probiotic strains and their mechanism of action involved in the removal of aflatoxin; and last section discusses the outcome of major studies showing aflatoxin reduction potential of food grade bacteria in milk and milk based products.

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Several toxic metabolites, such as aflatoxin M1 (AFM1), are known to contaminate dairy milk. However, as mentioned in an external EFSA report, there is a knowledge gap regarding the carry-over of certain emerging toxins such as microcystin-LR (MC-LR). Therefore, this work aimed to develop an LC-MS/MS method for MC-LR quantification in dairy milk. Also, the method included AFM1 as a common fungal metabolite and applied to analyze 113 dairy milk samples collected directly after the end of the summer peak. Both toxins were below their LODs, keeping the question on MC-LR carry-over still unanswered. Moreover, an in silico analysis, using a 3D molecular modeling was performed, pointing to a possible interaction between MC-LR and milk proteins, especially ß-lactoglobulin. Since AFM1 and MC-LR are hepatotoxic, their interaction in inducing mitochondrial dysfunction in HepG2 cells was investigated at low (subcytotoxic) concentrations. Live cell imaging-based assays showed an inhibition in cell viability, without involvement of caspase-3/7, and a hyperpolarization in the mitochondrial membrane potential after the exposure to a mixture of 100 ng mL-1 AFM1 and 1000 ng mL-1 MC-LR for 48h. Extracellular flux analysis revealed inhibitions of several key parameters of mitochondrial function (basal respiration, ATP-linked respiration, and spare respiratory capacity).

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The detection of the amount of aflatoxin M1 (AFM1) in milk is crucial for food safety. Here, we utilize a fiber optic (FO) localized surface plasmon resonance (LSPR) biosensor by constructing gold nanoparticle (AuNP) multimers, in which the nanogaps amplified the LSPR signal by the hot spot effect, and achieved a highly sensitive detection of f AFM1. Through the optimization of parameter conditions for the fabrication of the sensor and detection system, a high performance result from the FO LSPR biosensor was obtained, and the method for AFM1 detection was established, with a wide detection range of 0.05-100 ng/mL and a low limit of detection (LOD) of 0.04 ng/mL, and it has been successfully validated with the actual sample milk. Therefore, it is a good strategy to fabricate highly sensitive FO LSPR sensors for detecting AFM1 by constructing AuNP multimers, and this approach is suitable for developing other biosensors.

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Aflatoxin B1 (AFB1), a common mycotoxin, can occur in agricultural products. As a metabolite of AFB1, aflatoxin M1 (AFM1) mainly exist in dairy products. These two mycotoxins threaten human health, although it is unclear how they affect the function of the intestinal barrier. In this study, mice were exposed to AFB1 (0.3 mg/kg body b.w.) and AFM1(3.0 mg/kg b.w.) either individually or in combination for 28 days to explore the main differentially expressed proteins (DEPs) and the associated enriched pathways. These findings were preliminarily verified by the transcriptomic and proteomic analyses in differentiated Caco-2 cells. The results revealed that AFB1 and AFM1 exposure in mice disrupted the function of the intestinal barrier, and the combined toxicity was greater than that of each toxin alone. Further proteomic analysis in mice demonstrated that the mechanisms underlying these differences could be explained as follows: (i) lipid metabolism was enriched by AFB1-induced DEPs. (ii) protein export pathway was stimulated by AFM1-induced DEPs. (iii) cell metabolic ability was inhibited (as evidenced by changes in UDP-GT1, UDP-GT2, and Gatm6), apoptosis was induced (MAP4K3), and epithelial cell integrity was disrupted (Claudin7 and IQGAP2), resulting in more extensive intestinal damage after combined treatment. In conclusion, the hazardous impact of co-exposure to AFB1 and AFM1 from proteomic perspectives was demonstrated in the present study.

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Background: Fungal metabolites known as aflatoxin M1 (AFM1) are linked to contaminated milk and milk products. Consuming food contaminated with AFM1 poses major health risks and may even be fatal. Methods: The retrieved publications were categorized in this bibliometric study using the Web of Science (WoS) database Jan 1, 1970 to Nov 30, 2022 based on a variety of factors, including the time of publication of articles, citation totals, languages, research areas, countries, affiliations, funding agencies, journals, and keywords analysis to identify any hot and developing subjects. Additionally, VOSviewer software version 1.6.18 provided the bibliometric analysis of the global collaboration network and hot research themes. Results: Overall, 679 published documents were detected. Food Control was the top-line journal in publications on AFM1 research with 540 published articles, while the USA was the best productive country in AFM1 publications as well as the major country with the maximum co-authorship collaboration. This study ensures quantitative and qualitative analyses of the top 25 journals, most cited published articles, most relevant authors and title word occurrences in published documents on AFM1 publications. Over the past two decades, there has been an enormous rise for research conducted on global AFM1. Conclusion: The assessment of the historical state and development trend in AFM1 scientific research can serve as a roadmap for future research and eventually, serve as a foundation for bettering management practices for territorial decisions, healthcare, and dairy industries.

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This paper presents the first assessment of dietary exposure to aflatoxin M1 (AFM1) and associated health risks through milk and dairy product consumption in Armenia. Data on AFM1 in raw milk were obtained from an annual residue monitoring program. Additionally, commonly consumed dairy products (pasteurized milk, cheese, sour cream, curd cheese) were sampled, considering the sources of raw milk used by dairy companies. Per capita consumption of raw milk was sourced from national food balance databases, while individual consumption data for dairy products was collected via a 24 h recall survey with 1400 adult respondents. Detectable levels of AFM1 were observed in 7.14% of raw milk samples (up to 0.334 µg/kg) and, albeit at lower amounts (up to 0.009 µg/kg), in 30% and 40% of sour cream and curd cheese, respectively. The AFM1 levels were lower than the national maximum permitted level (0.5 µg/kg); however, levels in raw milk exceeded the EU ML (0.05 µg/kg). The estimated margin of exposure values for dairy products indicated no significant risk, whereas a reasonable worst-case estimate, using the measurable levels of AFM1 in raw milk consumption indicated a potential public health concern. This study provides a scientific basis for evaluating aflatoxin issues in the Caucasus area.

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Background: The assessment of risks related to food safety is becoming a challenge in developing countries with its consequent health hazards. Chemical risk assessment in dairy products is important to maintain consumer health locally and internationally. Since milk and dairy products are essential foods for a wide range of customers, mostly children, patients, and pregnant women, it is very important to estimate the risks of some chemical residues, such as pesticides, some heavy metals, and aflatoxins. Aim: This work aims to determine the levels of chemical contamination in milk and traditional Egyptian cheese. Methods: Heavy metals were determined in samples by atomic absorption spectrometry. GC-mass spectrometry (MS)/MS and LC-MS/MS were also used for measuring pesticide residues. The Aflatoxin M1 was determined by enzyme-linked immune-sorbent assay. Results: Raw milk samples were tested and showed elevated concentrations of lead and cadmium, (46% and 4%, respectively). The heavy metals detected in the Egyptian cheese samples were variable depending on the type of cheese. Moreover, p.p.-DDE phenofose was present in 45% and 29% of raw milk and Ras cheese samples, respectively. For Aflatoxin M1, only 7% of milk samples and 2.9% of Ras cheese samples exceeded the acceptable limits. Conclusion: More surveying and risk assessment of chemical residues in milk and milk products are essential for controlling health risks to consumers.

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Wagashi is a West African type cottage cheese locally prepared from cow milk. Wagashi like other milk products, is prone to microbial contamination, particularly by fungi. Many of these fungal species produce mycotoxins which are of serious public health concern. This work aimed to update the mycoflora profile and determine the concentrations of aflatoxin M1 and its health risk characterization due to the consumption of wagashi. Culturing the wagashi on mycological media (Oxytetracycline Glucose Yeast Extract OGYE, Dichloran Rose Bengal Chloramphenicol DRBC) caused a de-novo growth of the quiescent spores at 28-30 °C for 5-7 days. The analysis of AFM1 levels in the samples was done using High-Performance Liquid Chromatography connected to a Fluorescence detector (HPLC-FLD). The exposure and risk assessment to the AFMI levels were determined using deterministic models prescribed by the European Food Safety Authority (EFSA). The fungal counts ranged between 2.36-4.30 log10 CFU/g. In total, thirteen (13) fungal species from eight (8) genera were isolated from all wagashi samples. They are; Fusarium oxysporum, Aspergillus flavus, Aspergillus niger, Fusarium verticillioides, Penicillium digitatum, Trichoderma harzianum, Aspergillus terreus, Rhodotorula mucilaginosa, Rhizopus stolonifer, Aspergillus fumigatus, Yeast sp., Mucor racemosus and Fusarium oligosporum belonging to the genera Fusarium, Aspergillus, Penicillium, Trichoderma, Rhodotorula, Rhizopus, Yeast, and Mucor. The AFM1 observed in the wagashi samples' analysis was low, ranging from 0.00 (Not Detected) ± 0.00 - 0.06 ± 0.002 µg/Kg. Risk assessments of AFM1 using deterministic models produced outcomes that ranged between 5.92 × 10-3- 0.14 ng/kg bw/day, 1.42 -44.35, 0-0.0323 ng aflatoxins/kg bw/day, and 1.51 × 10-3 - 9.69 × 10-4 cases/100,000 person/yr for estimated daily intake (EDI), margin of exposure (MOE), average potency, and cancer risks, respectively, for the age categories investigated. Fungal counts were interpreted as medium to high. It was also established that the consumption of wagashi may pose adverse health effects on all age categories in the selected zones of the study since all calculated MOE values were less than 100,000.

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Given the highly mutagenic and carcinogenic nature of Aflatoxin M1 (AFM1), the quantity assessment of AFM1 residues in milk and dairy products is necessary to maintain consumer health and food safety. Herein, CRISPR-Cas12a-based colorimetric aptasensor was developed using the catalytic activity of flower-like nanozymes of MnO2 and trans-cleavage property of CRISPR-Cas12a system to quantitatively detect AFM1. The basis of the developed colorimetric aptasensor relies on whether or not the CRISPR-Cas12a system is activated, as well as the contrast in oxidase-mimicking capability exhibited by flower-like MnO2 nanozymes when AFM1 is absent or present. When AFM1 is not present in the sample, single-stranded DNA (ssDNA) is degraded by the activated CRISPR-Cas12a, and the solution turns into yellow due to the catalytic activity of the nanozymes. While, in the attendance of AFM1, ssDNA degradation does not occur due to the inactivation of the CRISPR-Cas12a. Therefore, with the adsorption of the ssDNA on the MnO2 nanozymes, their catalytic activity decreases, and the solution color becomes pale yellow due to less oxidation of the chromogenic substrate. In this aptasensor, the relative absorbance changes increased linearly from 6 to 160 ng L-1, and the detection limit was 2.1 ng L-1. The developed aptasensor displays a selective detection performance and a practical application for quantitative analysis of AFM1 in milk samples. The results of the introduced aptasensor open up the way to design other selective and sensitive aptasensors for the detection of other mycotoxins by substitution of the used sequences.

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Aflatoxin B1 (AFB1), a naturally-occurring mycotoxin, can cause severe toxicological and carcinogenic effects in livestock and humans. Given that the chicken is one of the most important food-producing animals, knowledge regarding AFB1 metabolism and enzymes responsible for AFB1 transformation in the chicken has important implications for chicken production and food safety. Previously, we have successfully expressed chicken CYP1A5 and CYP3A37 monooxygenases in E. coli, and reconstituted them into a functional CYP system consisting of CYP1A5 or CYP3A37, CPR and cytochrome b5. In this study, we aimed to investigate the roles of CYP1A5 and CYP3A37 in the bioconversion of AFB1 to AFM1. Our results showed that chicken CYP1A5 was able to hydroxylate AFB1 to AFM1. The formation of AFM1 followed the typical Michaelis-Menten kinetics. The kinetics parameters of Vmax and Km were determined as 0.83 ± 0.039 nmol/min/nmol P450 and 26.9 ± 4.52 µM respectively. Docking simulations further revealed that AFB1 adopts a "side-on" conformation in chicken CYP1A5, facilitating the hydroxylation of the C9a atom and the production of AFM1. On the other hand, AFB1 assumes a "face-on" conformation in chicken CYP3A37, leading to the displacement of the C9a atom from the heme iron and explaining the lack of AFM1 hydroxylation activity. The results demonstrate that chicken CYP1A5 possesses efficient hydroxylase activity towards AFB1 to form AFM1.

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Breast milk (BM) is considered as the best source of nutrition which could have prevention effects on various diseases in the first years of a child. Along with nutritive compounds, presence of contaminants such as mycotoxins in BM could be transmitted into neonate. The aim of this study was to determine the occurrence, levels, and factors associated with the presence of aflatoxin M1 (AFM1) and ocratoxin a (OTA) in BM samples of nursing mothers in rural centers of Yazd, Iran. The presence and average AFM1 and OTA concentration in 72 BM samples was measured by competitive ELISA. The demographic and diet parameters of nursing mothers were collected by a questionnaire and were analyzed using SPSS 18 software. AFM1 and OTA were detected in 63 (87.5%) and 47 (65.2%) samples with the mean concentration levels of 19.46 ± 13.26 ng/L (ranges from 5.1 to 53.9) and 200 ± 160 ng/L (ranges from 100 to 2460), respectively. Of these, 32 samples (50.7%) for AFM1 and 23 samples (48.9%) for OTA had values exceeding the limit set by the European Union regulation for infant foods (25 ng/L for AFM1 and 500 ng/L for OTA). It was also found that the risk of AFM1 and OTA occurrence in BM increased significantly with the consumption of beans, bread, cereals, fruit juice and crackers, and cream, respectively. This study showed that the estimated daily intake for AFM1 and OTA by 1 month of age infants was 2.7 and 28.5 ng/kg bw/day, respectively, while, as the age of the infant increased, the values were lower and close to 0.9 and 9.9 ng/kg bw/day for AFM1 and OTA in 12 months of age infants, respectively. The high occurrence and noticeable levels of AFM1 and OTA detected in this study indicated that some infants receive undesirable exposures to AFM1 and OTA with breast milk. Therefore, it is recommended that mothers are advised to avoid certain foods during pregnancy and breastfeeding that are likely sources of mycotoxins.

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AIM AND BACKGROUND: Aflatoxins, produced by Aspergillus flavus and Aspergillus parasiticus, are among the most toxic mycotoxins. Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1), found in milk and dairy products from animals fed AFB1-contaminated feed. Consumption of AFM1 has related adverse effects on human health. Breast milk can be a source of contamination for infants due to the presence of AFM. AFM1 can also contaminate powdered milk, a significant product of the milk industry. Consequently, monitoring dairy products for these toxins is imperative. STUDY METHOD: A total of 50 samples (25 samples of breast milk and 25 samples of powdered infant milk formula) were collected in Tehran from December 2021 to February 2022. HPLC method was used for the determination of AFM1 in samples. RESULTS: and Discussion: AFM1 was detected in 72% of breast milk samples and 96% of powdered milk samples. AFM1 levels varied significantly between the two sample types (p < 0.05). The average amount of AFM1 in breast milk samples was 25.82 ± 4.72 ng/kg, while the average amount in powdered milk samples was 40.59 ± 7.76 ng/kg. Moreover, 44% of the breast and 68% of powdered milk samples exceeded the AFM1 content limit of the European Union and the Iranian national standard. This study concludes that given the importance of breast milk and formula to maternal and infant health, monitoring and regulating the toxin levels in these products in Tehran is crucial.

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In view of the feed/foods inevitably contaminated by toxic and carcinogenic aflatoxin B1 (AFB1), efficient mesoporous metformin-chitosan/silica­cobalt ferrite nanospheres (Mt-CS/CFS NSs) was prepared to remove AFB1 from aqueous/non-aqueous media. The morphological, functional, and structural characteristics and adsorption properties of C/N-enriched CS/CFS were investigated systematically. The interactive operating variables (temperature (5.0-35 °C); time (10-100 min); AFB1 dose (50-100 µg/mL); and Mt-CS/CFS dosage (0.5-3.5 mg) were optimized via the Box-Behnken design (BBD), which demonstrated good agreement between the experimental data and proposed model. The adsorption efficiency in artificially contaminated cow's milk as well as aqueous environment reached over 91.0 % in a wide pH range (3.0-9.0), without significant change in the nutritional value of milk. Freundlich isotherm and second-order adsorption kinetics were regarded as the most suitable models to fit the adsorption results, and the adsorption rate is dominated by the intra-particle diffusion and boundary layer diffusion. Thermodynamic analyses proved that the process was spontaneous and exothermic. The adsorption mechanism could be explained as physisorption via hydrogen bonding, n-π interaction, and hydrophobic/hydrophilic interactions. The porous Mt-CS/CFS NS derived from chitosan nanoparticles is therefore outstanding adsorbent, offering great adsorptive performance and recycabilities, which impedes economic losses in the food industry.

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CONTEXT: Aflatoxins are the most harmful mycotoxins that cause human and animal health concerns. Aflatoxin M1 (AFM1) is the primary hydroxylated metabolite of aflatoxin B1 and is linked to the development of hepatocellular carcinoma and immunotoxicity in humans and animals. Because of the important role of dairy products in human life, especially children, AFM1 is such a major concern to humans because of its frequent occurrence in dairy products at concentrations high enough to cause adverse effects to human and animal health. Reduced its bioavailability becomes a high priority in order to protect human and animal health. OBJECTIVES: This study aimed to investigate, in vivo, the ability of lactic acid bacteria (lactobacillus rhamnosus GAF01, LR) and clay mineral (bentonite, BT) mixture to mitigate/reduce AFM1-induced immunotoxicity, hepatotoxicity, nephrotoxicity and oxidative stress in exposed Balb/c mice. MATERIALS AND METHODS: The in vivo study was conducted using male Balb/c mice that treated, orally, by AFM1 alone or in combination with LR and/or BT, daily for 10 days as follows: group 1 control received 200 µl of PBS, group 2 treated with LR alone (2.108 CFU/mL), group 3 treated with BT alone (1 g/kg bw), group 4 treated with AFM1 alone (100 µg/kg), group 5 co-treated with LR + AFM1, group 6 co-treated with BT + AFM1, group 7 co-treated with BT + LR + AFM1. Forty-eight h after the end of the treatment, the mice were sacrificed and the blood, spleen, thymus, liver and kidney were collected. The blood was used for biochemical and immunological study. Spleen and thymus samples were used to thymocytes and splenocytes assessments. Liver and kidney samples were the target for evaluation of oxidative stress enzymes status and for histological assays. RESULTS: The results showed that AFM1 caused toxicities in male Blab/c mice at different levels. Treatment with AFM1 resulted in severe stress of liver and kidney organs indicated by a significant change in the biochemical and immunological parameters, histopathology as well as a disorder in the profile of oxidative stress enzymes levels. Also, it was demonstrated that AFM1 caused toxicities in thymus and spleen organs. The co-treatment with LR and/or BT significantly improved the hepatic and renal tissues, regulated antioxidant enzyme activities, spleen and thymus viability and biochemical and immunological parameters. LR and BT alone showed to be safe during the treatment. CONCLUSION: In summary, the LR and/or BT was able to reduce the biochemical, histopathological and immunological damages induced by AFM1 and indeed it could be exploited as one of the biological strategies for food and feedstuffs detoxification.

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A novel ratiometric electrochemical aptasensor based on split aptamer and Au-reduced graphene oxide (Au-rGO) nanomaterials was proposed to detect aflatoxin M1 (AFM1). In this work, Au-rGO nanomaterials were coated on the electrode through the electrodeposition method to increase the aptamer enrichment. We split the aptamer of AFM1 into 2 sequences (S1 and S2), where S1 was immobilized on the electrode due to the Au-S bond, and S2 was tagged with methylene blue (MB) and acted as a response signal. A complementary strand to S1 (CS1) labeled with ferrocene (Fc) was introduced as another reporter. In the presence of AFM1, CS1 was released from the electrode surface due to the formation of the S1-AFM1-S2 complex, leading to a decrease in Fc and an increase in the MB signal. The developed ratiometric aptasensor exhibited a linear range of 0.03 µg L-1 to 2.00 µg L-1, with a detection limit of 0.015 µg L-1 for AFM1 detection. The ratiometric aptasensor also showed a linear relationship from 0.2 µg L-1 to 1.00 µg L-1, with a detection limit of 0.05 µg L-1 in natural milk after sample pretreatment, indicating the successful application of the developed ratiometric aptasensor. Our proposed strategy provides a new way to construct aptasensors with high sensitivity and selectivity.

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This study aimed to investigate Aflatoxin-M1 (AFM1) contamination in pasteurized and raw milk samples consumed in Kerman and Rafsanjan in southeastern Iran. In this cross-sectional study, a total of 100 samples of raw (n = 67) and pasteurized (n = 33) milk were randomly collected from retail stores, supermarkets, and milk transport tankers in the winter of 2020 and the summer of 2021. The level of AFM1 contamination in the collected samples was evaluated by high-performance liquid chromatography with fluorescence detection (HPLC-FD). AFM1 was detected in 95% of samples and its median concentration was 17.38 ng/L. The median concentration of AFM1 in the pasteurized milk samples (24.89 ng/L) was significantly higher than in the raw milk samples (13.54 ng/L). The AFM1 contamination level in 20% (raw = 13% and pasteurized = 7%) of the samples was higher than the maximum permitted level (MPL) recommended by the European Union (i.e., 50 ng/L), whilst 4% (raw = 3% and pasteurized = 1%) of the samples was higher than the Iranian maximum standard limit (i.e., 100 ng/L). The hazard index (HI) was higher than 1 in 16%, 18%, and 35% of total milk samples for men, women, and children, respectively. The AFM1 contamination level in the milk samples collected in southeastern Iran was worrying. The margin of exposure (MoE) values were lower than 10,000 for children. Because aflatoxins are among the most potent carcinogens known, prevention of milk contamination in all stages from the farm to the table can considerably reduce the community's exposure to AFM1 and its consequent health risks.

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Cheese is popular in Iran because of its high nutritional value; therefore, it is necessary to control this product regarding health risk factors, particularly aflatoxin M1 (AFM1). This research reviewed AFM1 in various varieties of cheese in Iran to assess the potential health risks associated with consuming these products for different age groups. In this regard, all accessible papers from different databases were screened between June 27, 2000 and October 10, 2022 b y systematic research and then considering the selection criteria of the studies; finally, 22 articles were selected for the current review. The amount and prevalence of AFM1 were calculated and separated based on the cheese variety, and the sampling location; health risk assessment (HRA), statistical, uncertainty, and sensitivity analysis for AFM1 of cheese for different age groups were performed. The study results for 2143 samples showed that the overall average AFM1 for cheese is 160 ± 175 ng/kg, below the European Commission (EC) regulation (250 ng/kg). AFM1 contaminated 72.42% of all cheese samples, and 13% of these contaminated samples had a higher AFM1 than the EC regulation. Cheese varieties were ranked based on average levels of AFM1 as white pasteurized > traditional > creamy > probiotic > Lighvan, and this ranking was obtained based on sampling locations as market > dairy factories > livestock farms. Based on the HRAs, from the perspective of the liver cancer risk (LCR), the margin of exposure (MOE), and the hazard index (HI) approach, it can be concluded that cheese produced in Iran, in terms of AFM1, particularly for children, poses serious health risks. Accordingly, it is imperative to carefully consider implementing suitable management methods to inhibit the growth of aflatoxin B1 (AFB1) in livestock fodder, and training in sanitary production and processing of dairy products according to world standards is suggested for industrial and traditional cheese producers across Iran.