Aflatoxin M1 Research Articles

Superoxide dismutase, a novel aflatoxin oxidase from Bacillus pumilus E-1-1-1: Study on the degradation mechanism of aflatoxin M1 and its application in milk and beer

The aim of this work was to study the effect of a recombinant superoxide dismutase (rSOD) from Bacillus pumilus E−1-1-1 regarding the degradation of aflatoxin M1 (AFM1) in model solution. The degradation mechanism was further explored and applied in milk and beer. The Kinetic Momentum and maximal velocity of the rSOD towards AFM1 were 3.6 μg/mL and 1.8 μg/mL/min, respectively. The rSOD-mediated AFM1 degradation product was identified as C12H11O3. Molecular docking simulation suggested that hydrogen bonds, van der Waals interaction, and pi bond played major roles in the steadiness of AFM1-rSOD. Meanwhile, compared with identical concentration of AFM1, the survival rate of Hep-G2 cells treated with superoxide dismutase-generated AFM1 degradation products increased about 1.6 times. In addition, the degradation rates in lager beer and milk were 15.19% and 26.03%, respectively. Therefore, the rSOD has great modification value to accommodate AFM1 degradation in beer and milk. This study indicates a direction to be followed for future degrading AFM1 in food.

Probabilistic Health Risk Assessment of Aflatoxin M1 in Pasteurized Milk from Different Cities of Iran

Probabilistic Health Risk Assessment of Aflatoxin M1 in Pasteurized Milk from Different Cities of Iran

Determination of aflatoxin M1 and ochratoxin A in breast milk in rural centers of Yazd, Iran: Exposure assessment and risk characterization.

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 ± 13.26ng/L (ranges from 5.1 to 53.9) and 200 ± 160ng/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 (25ng/L for AFM1 and 500ng/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 1month of age infants was 2.7 and 28.5ng/kg bw/day, respectively, while, as the age of the infant increased, the values were lower and close to 0.9 and 9.9ng/kg bw/day for AFM1 and OTA in 12months 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.

CRISPR-Cas12a-based colorimetric aptasensor for aflatoxin M1 detection based on oxidase-mimicking activity of flower-like MnO2 nanozymes

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.

Biomonitoring of Dietary Mycotoxin Exposure and Associated Impact on the Gut Microbiome in Nigerian Infants.

Mycotoxins are toxic chemicals that adversely affect human health. Here, we assessed the influence of mycotoxin exposure on the longitudinal development of early life intestinal microbiota of Nigerian neonates and infants (NIs). Human biomonitoring assays based on liquid chromatography tandem mass spectrometry were applied to quantify mycotoxins in breast milk (n = 68) consumed by the NIs, their stool (n = 82), and urine samples (n = 15), which were collected longitudinally from month 1-18 postdelivery. Microbial community composition was characterized by 16S rRNA gene amplicon sequencing of stool samples and was correlated to mycotoxin exposure patterns. Fumonisin B1 (FB1), FB2, and alternariol monomethyl ether (AME) were frequently quantified in stool samples between months 6 and 18. Aflatoxin M1 (AFM1), AME, and citrinin were quantified in breast milk samples at low concentrations. AFM1, FB1, and ochratoxin A were quantified in urine samples at relatively high concentrations. Klebsiella and Escherichia/Shigella were dominant in very early life stool samples (month 1), whereas Bifidobacterium was dominant between months 3 and 6. The total mycotoxin levels in stool were significantly associated with NIs' gut microbiome composition (PERMANOVA, p < 0.05). However, no significant correlation was observed between specific microbiota and the detection of certain mycotoxins. Albeit a small cohort, this study demonstrates that mycotoxins may influence early life gut microbiome composition.

Chicken CYP1A5 is able to hydroxylate aflatoxin B1 to aflatoxin M1

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.

Detection of Aflatoxin M1 and Several Heavy Metals in Medical Infant Milk Formula Sold in Iraqi Markets

Milk is essential for infants, and infant feeding should be emphasized in any program to ensure good child health. To find harmful and trace elements in different milk powder brands bought in Iraq and fed to babies with metabolic syndrome, atomic absorption spectrometry (AAS) was used. Fluorescence high-performance liquid chromatography (HPLC) was also used to measure aflatoxin M1. The result reveals the concentrations of elements close to the product label's description, such as magnesium, calcium, zinc, selenium, iron, and manganese. Only the potassium element was higher than what was listed on the product label. Additionally, no quantities of hazardous elements like lead (Pb), cadmium (Cd), or arsenic (As) were found. As for aflatoxin M1, it was found that the PKU milk formula and lactose-free milk formula were found to be free of AFM1 contamination, indicating that they are safe to consume. AFM1 was found in concentrations above the permissible limit in the MSUD milk formula (0.08 µg/kg). The reason for this issue could be inadequate storage. The other two types, TYR and OAc milk formula, were lower than the allowable limit of 0.05 µg/kg. As a result, there needs to be more oversight of the milk that newborns with metabolic syndrome consumele S. aureus or Streptococcus pyogenes, and Streptococcus pneumoniae in community settings.

Fungal profile, levels of aflatoxin M1, exposure, and the risk characterization of local cheese ‘wagashi’ consumed in the Ho Municipality, Volta Region, Ghana

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.

Trends in Aflatoxin M1 Global Research: A Bibliometric Analysis Study.

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. 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. 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. 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.

Aflatoxin M1 Analysis in Urine of Mill Workers in Bangladesh: A Pilot Study.

Presence of aflatoxin B1 (AFB1) in food and feed is a serious problem, especially in developing countries. Human exposure to this carcinogenic mycotoxin can occur through dietary intake, but also through inhalation or dermal contact when handling and processing AFB1-contaminated crops. A suitable biomarker of AFB1 exposure by all routes is the occurrence of its hydroxylated metabolite aflatoxin M1 (AFM1) in urine. To assess mycotoxin exposure in mill workers in Bangladesh, we analyzed AFM1 levels in urine samples of this population group who may encounter both dietary and occupational AFB1 exposure. In this pilot study, a total of 76 participants (51 mill workers and 25 controls) were enrolled from the Sylhet region of Bangladesh. Urine samples were collected from people who worked in rice, wheat, maize and spice mills and from controls with no occupational contact to these materials. A questionnaire was used to collect information on basic characteristics and normal food habits of all participants. Levels of AFM1 in the urine samples were determined by a competitive enzyme linked immunosorbent assay. AFM1 was detected in 96.1% of mill workers' urine samples with a range of LOD (40) of 217.7 pg/mL and also in 92% of control subject's urine samples with a range of LOD of 307.0 pg/mL). The mean level of AFM1 in mill workers' urine (106.5 ± 35.0 pg/mL) was slightly lower than that of the control group (123.3 ± 52.4 pg/mL), whilst the mean AFM1 urinary level adjusted for creatinine was higher in mill workers (142.1 ± 126.1 pg/mg crea) than in the control group (98.5 ± 71.2 pg/mg crea). Yet, these differences in biomarker levels were not statistically significant. Slightly different mean urinary AFM1 levels were observed between maize mill, spice mill, rice mill, and wheat mill workers, yet biomarker values are based on a small number of individuals in these subgroups. No significant correlations were found between the study subjects' urine AFM1 levels and their consumption of some staple food items, except for a significant correlation observed between urinary biomarker levels and consumption of groundnuts. In conclusion, this pilot study revealed the frequent presence of AFM1 in the urine of mill workers in Bangladesh and those of concurrent controls with dietary AFB1 exposure only. The absence of a statistical difference in mean biomarker levels for workers and controls suggests that in the specific setting, no extra occupational exposure occurred. Yet, the high prevalence of non-negligible AFM1 levels in the collected urines encourage further studies in Bangladesh regarding aflatoxin exposure.

Evaluation of aflatoxin M1 and nutritional content in Kosovo’s market cheese samples

The ubiquitous presence and potent carcinogenic effects of aflatoxins, a group of mycotoxins, pose a significant threat to global food safety, especially within the dairy sector. This research aimed to evaluate the prevalence and distribution of Aflatoxin M1 in diverse cheese varieties within Kosovo’s markets in 2022 and discern potential correlations with cheese types, processing conditions, and nutritional parameters to enhance understanding and ensure the safety of dairy products. In the latter half of 2022, a total of 93 different types of manufactured cheese samples from 15 different countries were analysed for Aflatoxin M1 toxin content using the enzyme-linked immunosorbent assay technique. The results of the research indicate that several countries have exceeded the concentration of 0.05 ug/kg for Aflatoxin M1, set as the maximum tolerable limit for milk and milk products in many countries. Notable, cheese samples from Albania exhibited substantially (p=0.000) higher levels of Aflatoxin M1 compared to other nations. The results showed that the Aflatoxin M1 concentration did not exhibit statistically significant variations based on the regulated total fat content (p=0.902), carbohydrate content (p=0.741), protein concentration (p=0.298), or salt content (p=0.122) within the cheese. The results demonstrated that Aflatoxin M1 levels were not significantly (p=0.926) affected by cheese consistency. While different starter cultures were associated with varying Aflatoxin M1 concentrations, these differences were not statistically significant (p=0.472). Similarly, ageing time did not exert a significant (p=0.691) impact on AFM1 levels, as concentrations remained relatively consistent across different ageing periods. These findings provide valuable insights into the variations in Aflatoxin M1 levels in cheese samples among different countries. They highlight the importance of regulatory measures and continuous monitoring to ensure food safety and adherence to maximum allowable limits

AFM1 exposure in male balb/c mice and intervention strategies against its immuno-physiological toxicity using clay mineral and lactic acid bacteria alone or in combination

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.

Density Functional Theory Study on the Interaction between Aflatoxin B1/M1 and Gold Substrate.

Aflatoxin M1 (AFM1) and its precursor, Aflatoxin B1 (AFB1), are highly pathogenic and mutagenic substances, making the detection and sensing of AFB1/M1 a long-standing focus of researchers. Among various detection techniques, surface-enhanced Raman spectroscopy (SERS) is considered an ideal method for AFB1/M1 detection due to its ability not only to enhance characteristic frequencies but also to detect shifts in these frequencies with high repeatability. Therefore, we employed density functional theory in conjunction with surface-enhanced Raman spectroscopy to investigate the interaction between AFB1/M1 and a Au substrate in the context of the SERS effect for the first time. To predict the potential binding sites of AFB1/M1 and Au within the SERS effect, we performed calculations on the molecular electrostatic potential of AFB1/M1. Considering the crucial role of the binding energy in molecular docking studies, we computed the binding energy between two molecules interacting with Au at different binding sites. The molecular frontier orbitals and related chemical parameters of AFB1/M1 and "molecular-Au" complexes were computed to elucidate the alterations in AFB1/M1 molecules under the SERS effect. Subsequently, the theoretical Raman spectra of AFB1/M1 and the complexes were compared and analyzed, enabling determination of the adsorption conformation of AFB1/M1 on the gold surface based on SERS surface selection rules. These findings not only provide a deeper understanding of the interaction mechanism between molecules and substrates in the SERS effect but also offer theoretical support for developing novel aflatoxin SERS sensors.

Aflatoxin M1 Concentrations, Adulterants, Microbial Loads, and Physicochemical Properties of Raw Milk Collected From Nekemte City, Ethiopia.

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.

Recent aflatoxin levels in maize, feed mixtures, milk and cheese in Serbia

Aflatoxins are the most widespread mycotoxins primar?ly produced by fungi of the genus Aspergillus such as A. flavus and A. parasiticus, infecting grains during storage, spices, nuts, milk and animal feed. Even in low concentrations, they are highly toxic. Major forms of aflatoxins include B1, B2, G1, G2, and M1. Mammals that ingest AFB1 contaminated food eliminate amounts of the main hepatic metabolite known as milk toxin or aflatoxin M1 (AFM1) via milk. The incidence of contamination of aflatoxin in maize, feed mixtures, milk and cheese samples collected from Serbian producers was investigated by using the competitive enzyme linked immunosorbent assay (ELISA) technique. In this study, a total of 22 samples of maize, 25 samples of feed mixtures, 284 samples of raw, pasteurised and UHT milk and 20 samples of cheese were examined in 2021. Aflatoxin B1 (AFB1) was quantified in 3 samples (13.6%) of maize, at levels ranging from 2.45?g/kg to 48.31?g/kg and 12 samples (48%) of feed mixtures at levels ranging from 2.04 ?g/kg to 21.48 ?g/kg. On the other hand, AFM1 was quantified in 51 samples (17.90%) of milk at levels ranging 0.02 ?g/kg to 0.26 ?g/kg and 15 samples (75%) of cheese at levels ranging from 0.15 ?g/kg to 0.46 ?g/kg. In the Republic of Serbia, maximum limit of AFB1 in maize used for livestock is 30 ?g/kg and for feed mixtures for dairy cows is 5 ?g/kg. On the other hand, maximum limit of AFM1 in milk is 0.25 ?g/kg. Maximum limit of AFM1 in cheese is not set. These results suggest the obvious presence of AFB1 in maize and the fact that number of feed mixture, milk and cheese samples contaminated with aflatoxins is not negligible. The occurence of aflatoxins in raw milk and comercially available milk is one of the most serious problems, as milk is a key source of nutrients for infants and young children. There?fore, continuous monitoring over milk is necessary as well as further research and risk analysis on AFM1 presence in cheese.

A field-portable electrochemical immunosensor based on a multifunctional Ag2O/g-C3N4@MA-DBB covalent organic framework receptor interface for single-step detection of aflatoxin M1 in raw milk samples.

Aflatoxin M1 (AFM1), a hydroxylated metabolite of aflatoxin B1 (AFB1), contaminates milk and dairy products from livestock via ingestion of feed contaminated with a species of Aspergillus. As AFM1 is a Group-II B carcinogen, it is indispensable to develop novel, easy-to-handle, sensitive, portable and cost-effective strategies for its detection. Herein, a covalent organic framework (COF)-based electroactive nanocomposite, Ag2O/g-C3N4-COOH@MA-DBB-COF (silver oxide/carboxy-functionalized graphitic carbon nitride@melamine-dibromo butane COF), is designed to serve as a multifunctional receptor surface. The Ag2O/g-C3N4-COOH@MA-DBB-COF formation was characterized through X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, micro-Raman spectroscopy (RAMAN), dynamic light scattering (DLS) and thermogravimetric analysis (TGA), and each step of the sensor fabrication was monitored using field emission scanning electron microscopy (FE-SEM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Under the optimal conditions, the designed immunosensor permitted the detection of AFM1 in the linear range of 0.03-1000 fg mL-1, with a 0.01 fg mL-1 limit of detection (LOD). The selectivity of the designed immunosensor was validated via an anti-interference study. The practical applicability of the immunosensor was demonstrated by the detection of AFM1 in real milk samples, and good recovery values (97.28-102.62%) were obtained. Furthermore, the developed immunosensor and high-performance liquid chromatography (HPLC) were employed in parallel to detect AFM1 in local market milk samples from twenty different sites to validate the performance of the newly designed immunosensor. Additionally, the designed immunosensor was stable over an extended period of time. This work reports a single-step field-portable multifunctional innovative electrochemical immunoreceptor design for on-site and label-free detection of AFM1 in milk samples. Hence, the present study is the first report on the fabrication of a multifunctional innovative electrochemical immunoreceptor based on PGE/Ag2O/g-C3N4-COOH@MA-DBB-COF for the detection of AFM1 in milk samples.

Assessment of some chemical residues in Egyptian raw milk and traditional cheese.

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. This work aims to determine the levels of chemical contamination in milk and traditional Egyptian cheese. 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. 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. More surveying and risk assessment of chemical residues in milk and milk products are essential for controlling health risks to consumers.

Degradation of Aflatoxin M1 by Lipase and Protease in Buffer Solution and Yoghurt

Objective: This study aimed to examine the effectiveness of lipase and protease obtained from bacteria in the degradation of aflatoxin M1 (AFM1) in phosphate-buffered saline (PBS) and during the production of yoghurt. Methods: In this study, two strains, Levilactobacillus brevis and Lactobacillus plantarum, were used to produce protease and lipase, respectively. We then investigated the ability of protease and lipase to degrade AFM1 at four concentrations (50, 100, 150, and 200 U/ml for each enzyme) in vitro and during the preparation of yoghurt. Results: The results revealed that the highest activity was recorded at pH 7 and 7.5 for protease and lipase, respectively. As well, the optimum activity was observed at temperatures of 50 °C and 30 °C for protease and lipase, respectively. In vitro, the lipase enzyme at 200 U/ml degraded the AFM1 to 31.8, 37.4, and 56.7%, after incubating the PBS for 6, 12, and 18 h, respectively. Concerning protease, the means of degradation for AFM1 were 35.03, 43.7, and 72.9%, under the same conditions in yoghurt made from samples contaminated with 10 μg/L of AFM1, which was treated by both lipase and protease enzymes at 0.3, 0.6, and 0.9%, respectively. In yoghurt made from contaminated milk at 10 μg/L for AFM1, which was treated by 0.3, 0.6, and 0.9% of both lipase and protease, after two days of storage, the means of degradation of AFM1 were 23.4, 37.8, and 65.9%, respectively, which increased after five days to 27.3, 52.6, and 78.5%, respectively. Conclusion: Degradation of AFM1 was examined during the manufacturing of yoghurt using both bacterial lipase and protease without significantly affecting the sensory qualities of the finished product. Because of this, these enzymes could be a useful option in the biotech and dairy industries.

Impact of ImarsilTM Adsorption on Aflatoxin M1 (AFM1) Levels in Cow's Milk: Analyzing Hematological Parameters and Histopathological Alterations

The efficacy of ImarsilTM in mitigating the effects of aflatoxin M1 (AFM1) in cow's milk on hematological and histopathological parameters was investigated in this study. Seventy-two albino rats were randomly allocated to four treatment groups A - D in a six-week study. Rats in all groups were fed standard ration. In addition, 2 mL of clean distilled water, 2 mL of milk, 2 mL of AFM1 contaminated milk (456 ng/L), and 2 mL of AFM1 contaminated milk (456 ng/L) treated with ImarsilTM at 2% dosage rate were added to the ration of animals in groups A, B, C, and D respectively. The results of the investigation showed that rats in Group C developed a significant (p&lt;0.05) lower weight. Packed Cell Volume (%), Hemoglobin (g/dL), Red Blood Cell (106/mm3), Mean Corpuscular Volume (fL), Mean Corpuscular Hemoglobin (pg), and Mean Corpuscular Hemoglobin Concentration (g/dL) were not significantly different (p&gt;0.05) among the different groups. In group C, a significant reduction (p&lt;0.05) occurred in the white blood cell (103/mm3) (12.90 - 8.63), and lymphocytes (87.00 - 74.33%) counts while the neutrophils (%) increased from 13.00 to 25.67. In contrast to those in Group C, tissue sections from Group D showed no histological lesions. Therefore, ImarsilTM represents an effective and safe adsorbent for the remediation of AFM1-contaminated milk.

Occurrence of aflatoxins in Nigerian foods

Aflatoxins are a family of poisonous, mutagenic, and carcinogenic mycotoxins that contaminate a wide range of foods and agricultural goods. Aspergillus species, such as Aspergillus flavus, and Aspergillus parasiticus are the most common producers. Aflatoxin generation can occur at any point of the food chain, including pre-harvest, drying, storage, transit, processing, and handling, if conditions are favourable for fungus to create toxins. It is classified into six main types which are Aflatoxin B1, Aflatoxin B2, Aflatoxin G1, Aflatoxin G2, Aflatoxin M1 and Aflatoxin M2. In Nigeria, Aspergillus species that produces aflatoxin has been isolated from agricultural products such as cereals, spices, locally fermented food, oil-seeds, and animal products. Aflatoxin contamination is high due to poor storage of food crops and lack of awareness of aflatoxins contamination among farmers, marketers and the consumers of these goods. Locally fermented foods such as ogiri, ugba, ogi-baba, and iru have been said to be contaminated by aflatoxin. Preventive measures should be carried out by the policy-making bodies to create awareness and sustain ongoing measures to effectively manage aflatoxin contamination in Nigeria so as to reduce the health risk of aflatoxins on the people and economy of the country.