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.

New insights into aflatoxin B1 mechanistic toxicology in cattle liver: an integrated approach using molecular docking and biological evaluation in CYP1A1 and CYP3A74 knockout BFH12 cell lines.

Aflatoxin B1 (AFB1) is a pro-carcinogenic compound bioactivated in the liver by cytochromes P450 (CYPs). In mammals, CYP1A and CYP3A are responsible for AFB1 metabolism, with the formation of the genotoxic carcinogens AFB1-8,9-epoxide and AFM1, and the detoxified metabolite AFQ1. Due to climate change, AFB1 cereals contamination arose in Europe. Thus, cattle, as other farm animals fed with grains (pig, sheep and broiler), are more likely exposed to AFB1 via feed with consequent release of AFM1 in milk, posing a great concern to human health. However, knowledge about bovine CYPs involved in AFB1 metabolism is still scanty. Therefore, CYP1A1- and CYP3A74-mediated molecular mechanisms of AFB1 hepatotoxicity were here dissected. Molecular docking of AFB1 into CYP1A1 model suggested AFB1 8,9-endo- and 8,9-exo-epoxide, and AFM1 formation, while docking of AFB1 into CYP3A74 pointed to AFB1 8,9-exo-epoxide and AFQ1 synthesis. To biologically confirm these predictions, CYP1A1 and CYP3A74 knockout (KO) BFH12 cell lines were exposed to AFB1. LC-MS/MS investigations showed the abolished production of AFM1 in CYP1A1 KO cells and the strong increase of parent AFB1 in CYP3A74 KO cells; the latter result, coupled to a decreased cytotoxicity, suggested the major role of CYP3A74 in AFB1 8,9-exo-epoxide formation. Finally, RNA-sequencing analysis indirectly proved lower AFB1-induced cytotoxic effects in engineered cells versus naïve ones. Overall, this study broadens the knowledge on AFB1 metabolism and hepatotoxicity in cattle, and it provides the weight of evidence that CYP1A1 and CYP3A74 inhibition might be exploited to reduce AFM1 and AFBO synthesis, AFB1 toxicity, and AFM1 milk excretion.

Ratiometric electrochemical aptasensor based on split aptamer and Au-rGO for detection of aflatoxin M1.

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.

Omics analysis revealed the intestinal toxicity induced by aflatoxin B1 and aflatoxin M1.

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.

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.

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.

[Dietary exposure assessment of aflatoxins of residents in Shanghai residents aged 15 years and above from 2018 to 2023].

OBJECTIVE: To understand the contents of aflatoxins(AFs) in foods sold in Shanghai, and to assess the exposure assessment of and its potential health risk among residents over 15 years old in Shanghai. METHODS: A total of 8114 samples from 8 categories of food were collected in Shanghai from 2018 to 2023. The samples were detected by GB 5009.24-2016 and GB 5009.22-2016. Combined with the food consumption data of "Shanghai Diet and Health Survey", the dietary exposure assessment of aflatoxin was conducted using the margin of exposure(MOE) and the risk of liver cancer. RESULTS: The detection rates of aflatoxin B_1(AFB_1), aflatoxin B_2(AFB_2), aflatoxin G_1(AFG_1), aflatoxin G_2(AFG_2), and aflatoxin M_(1 )(AFM_1) were 8.6%, 2.0%, 0.9%, 0.2% and 0.2%, respectively. The point assessment showed that the total exposure of AFB_1 in the diet of residents aged 15 and above in Shanghai was 0.783 ng/(kg·BW·d), with the contribution rates of dietary exposure to grains and their products, nuts and their products, and vegetable oils accounting for 60.6%, 25.0% and 8.5% of AFB_1's dietary exposure, respectively. The total exposure of total aflatoxins(the sum of AFB_1, AFB_2, AFG_1 and AFG_2)(AFT) was 7.363 ng/(kg·BW·d), and the dietary exposure of grains and their products, vegetable oils, nuts and their products contribute 77.1%, 8.4% and 7.2% to the dietary exposure of AFT, respectively. The probability assessment result indicated that the average dietary exposure of residents to AFB_1 and AFT were 0.734 and 7.220 ng/(kg·BW·d), respectively, and the P95 exposure of residents were 1.170 and 11.500 ng/(kg·BW·d). The AFB_1 exposure level of residents in suburban areas was higher than that in central urban areas and exurban areas(χ~2= 16.357, P<0.001), the AFT exposure of residents in the central urban area was lower than that in the exurban areas and suburban areas(χ~2= 40.996, P<0.001). According to the MOE method, the MOE values of AFB_1 and AFT average dietary exposure for residents aged 15 and above in Shanghai were 511 and 54. The risk of liver cancer caused by dietary exposure of AFB_1 and AFT among residents aged 15 and above in Shanghai were 0.024 cases/10~5 people and 0.227 cases/10~5 people. CONCLUSION: There is AFs contamination in food sold in Shanghai, and grains and their products, nuts and their products, and vegetable oils are the main sources of AFs exposure in the diet of residents aged 15 and above in Shanghai.

Novel Porphyrinic Covalent Organic Polymer with Polarity-Switchable Dual Wavelength for Accurate and Sensitive Photoelectrochemical Sensing.

Herein, we synthesized a novel porphyrinic covalent organic polymer (TPAPP-PTCA PCOP) for constructing a polarity-switchable dual-wavelength photoelectrochemical (PEC) biosensor with ferrocene (Fc) and hydrogen peroxide (H2O2) as regulator and amplifier simultaneously. Interestingly, this new PCOP possessed both n-type and p-type semiconductor characteristics, which thus enabled the appearance of a dual-polarity photocurrent at two different excitation wavelengths. Furthermore, Fc and H2O2 could readily switch the photocurrent of PCOP to the cathode and anode stemming from its efficient electron collection and donation function, respectively. Based on these, a PCOP-based PEC biosensor skillfully integrating dual wavelengths with reliable accuracy and polarity switch with high sensitivity was instituted. As a result, the developed PEC biosensor exhibited a low detection limit down to 0.089 pg mL-1 for the most powerful natural carcinogen aflatoxin M1 (AFM1) assay. Impressively, the target exhibited a completely opposite photocurrent difference to the interfering substances, and the linear correlation coefficient of the assay was improved compared to single-wavelength detection. The PEC sensing platform not only provided a basis for exploring multicharacteristic photoactive material but also innovatively developed the detection mode of the PEC biosensor.

Simultaneous Dual-Sensing Platform Based on Aptamer-Functionalized DNA Hydrogels for Visual and Fluorescence Detection of Chloramphenicol and Aflatoxin M1.

In this study, a chloramphenicol and aflatoxin M1 aptamer-functionalized DNA hydrogel was designed for the simultaneous detection of chloramphenicol and aflatoxin M1 for the first time. The acrydite-modified chloramphenicol aptamer sequence was used to synthesize the DNA hydrogel and for visual detection of chloramphenicol depending on the gel-to-sol transition of the target-responsive DNA hydrogel. The DNA hydrogel formulation was set as follows: 60% of each linear polyacrylamide-DNA conjugate and 40% of acrylamide and chloramphenicol aptamer/DNA strand-1 at a molar ratio of 1:1, and the lowest concentration of chloramphenicol leading to gel dissociation was 1.0 nM at 25 °C. Furthermore, the formalized aptamer-functionalized DNA hydrogel was used to detect aflatoxin M1 by measuring the recovery of the fluorescence signal that was quenched when the FAM-labeled aflatoxin M1 aptamer and BHQ1-labeled DNA strand-2 were hybridized to form a double-stranded DNA in the network of hydrogel. The detection platform was successfully applied to the detection of chloramphenicol and aflatoxin M1, both in aqueous solution and in milk. The aptamer-functionalized DNA hydrogel had detection (LOD) and quantification limits (LOQ) for aflatoxin M1 as 1.7 and 5.2 nM, respectively. Using two aptamer sequences with high affinity and specificity, the dual-sensing platform based on the DNA hydrogel achieved higher selectivity for chloramphenicol and aflatoxin M1, which demonstrated its potential as a reliable simultaneous detection platform against two different targets for monitoring food safety.

A multiple lateral flow immunoassay based on AuNP for the detection of 5 chemical contaminants in milk.

Melamine (MEL), enrofloxacin (ENR), sulfamethazine (SMZ), tetracycline (TC), and aflatoxin M1 (AFM1) are the main chemical contaminants in milk. It is necessary to detect these miscellaneous chemical contaminants in milk synchronously to ensure the safety of the milk. In this study, a multiple lateral flow immunoassay (LFIA) was developed for the detection of MEL, ENR, SMZ, TC, and AFM1 in milk. Under optimal experimental conditions, the cutoff values were 25 ng/mL for MEL, 1 ng/mL for ENR, 2.5 ng/mL for SMZ, 2.5 ng/mL for TC, and 0.25 ng/mL for AFM1 in milk samples. The limits of detection of LFIA were 0.173 ng/mL for MEL, 0.078 ng/mL for ENR, 0.059 ng/mL for SMZ, 0.082 ng/mL for TC, and 0.0064 ng/mL for AFM1. The recovery rates of LFIA in milk were 83.2-104.4% for MEL, 76.5-127.3% for ENR, 96.8-113.5% for SMZ, 107.1-166.6% for TC, and 93.5-130.3% for AFM1. The coefficients of variation were all less than 15%. As a whole, the developed multiple lateral flow immunoassay showed potential as a highly reliable and excellent tool for the rapid and sensitive screening of MEL, ENR, SMZ, TC, and AFM1 in milk.

Effects of chronic low-dose aflatoxin B1 exposure in lactating Florida dairy goats.

In the past few years there has been a growing trend in the prevalence of aflatoxins, attributable to climate change, in substances destined for animal feeding, together with an increase in dairy product consumption. These facts have triggered great concern in the scientific community over milk pollution by aflatoxin M1. Therefore, our study aimed to determine the transfer of aflatoxin B1 from the diet into milk as AFM1 in goats exposed to different concentrations of AFB1, and its possible effect on the production and serological parameters of this species. For this purpose, 18 goats in late lactation were divided into 3 groups (n = 6) and exposed to different daily doses of aflatoxin B1 (T1 = 120 µg; T2 = 60 µg, and control = 0 µg), during 31 d. Pure aflatoxin B1 was administered 6 h before each milking in an artificially contaminated pellet. The milk samples were taken individually in sequential samples. Milk yield and feed intake were recorded daily, and a blood sample was extracted on the last day of exposure. No aflatoxin M1 was detected, either in the samples taken before the first administration, or in the control group ones. The aflatoxin M1 concentration detected in the milk (T1 = 0.075 µg/kg; T2 = 0.035 µg/kg) increased significantly on a par with the amount of aflatoxin B1 ingested. The amount of aflatoxin B1 ingested did not have any influence on aflatoxin M1 carryover (T1 = 0.066% and T2 = 0.060%), these being considerably lower than those described in dairy goats. Thus, we concluded that the concentration of aflatoxin M1 in milk follows a linear relationship with respect to the aflatoxin B1 ingested, and that the aflatoxin M1 carryover was not affected by the administration of different aflatoxin B1 doses. Similarly, no significant changes in the production parameters after chronic exposure to aflatoxin B1 were observed, revealing a certain resistance of the goat to the possible effects of that aflatoxin.

A network meta-analysis on the efficacy of different mycotoxin binders to reduce aflatoxin M1 in milk after aflatoxin B1 challenge in dairy cows.

The objective of this network meta-analysis was to determine the efficacy of different mycotoxin binders (MTB) to reduce aflatoxin M1 (AFM1) in milk. A literature search was conducted to identify in vivo research papers from different databases. Inclusion criteria were in vivo, dairy cows, description of the MTB used, doses of MTB, aflatoxin inclusion in the diet, and concentration of AFM1 in milk. Twenty-eight papers with 131 data points were selected. Binders used in the studies were hydrated sodium calcium aluminosilicate (HSCAS), yeast cell wall (YCW), bentonite, and mixes of several MTB (MX). The response variables were AFM1 concentration, AFM1 reduction in milk, total AFM1 excreted in milk, and transfer of aflatoxin from feed to AFM1 in milk. Data were analyzed with CINeMA and GLIMMIX procedures with the WEIGHT statement of SAS (SAS Inst. Inc.). The AFM1 concentration in milk decreased for bentonite (0.3 µg/L ± 0.05; mean ± SE) and HSCAS (0.4 µg/L ± 0.12), and tended to decrease for MX (0.6 µg/L ± 0.13) but was similar for YCW (0.6 µg/L ± 0.12), compared with control (0.7 µg/L ± 0.12). The percentage reduction of AFM1 in milk was similar for all MTB and different from control with a range of reduction from 25% for YCW to 40% for bentonite. The excretion of AFM1 in milk was lower in YCW (5.3 µg/L ± 2.37), HSCAS (13.8 µg/L ± 3.31), and MX (17.1 µg/L ± 5.64), and not affected by bentonite (16.8 µg/L ± 3.33) compared with control (22.1 µg/L ± 5.33). The transfer of aflatoxin B1 from feed into AFM1 in milk was lowest in bentonite (0.6% ± 0.12), MX (1.04% ± 0.27), and HSCAS (1.04% ± 0.21), and not affected in YCW (1.4% ± 0.10), compared with control (1.7% ± 0.35). The meta-analysis results indicate that all MTB reduced the AFM1 transfer into milk, where bentonite had the highest capacity and YCW the lowest.

The coexistence of aflatoxin M1 and ochratoxin A induced intestinal barrier disruption via the regulation of key differentially expressed microRNAs and long non-coding RNAs in BALB/c mice.

Food safety can be seriously threatened by the existence of both aflatoxin M1 (AFM1) and ochratoxin A (OTA) in milk and corresponding products. The importance of intestine integrity in preserving human health is widely understood in vitro, but the fundamental processes by which AFM1 and OTA cause disruption of the intestinal barrier are as yet unknown, especially in vivo. Based on the analysis of the whole transcriptome of BALB/c mice, the competing endogenous RNA (ceRNA) regulation network was obtained in the current study. Each of 12 mice were separated into five treatments: saline solution treatment, 1.0% DMSO vehicle control treatment, 3.0 mg/kg b.w. individual AFM1 treatment (AFM1), 3.0 mg/kg b.w. individual OTA treatment (OTA), and combined mycotoxins treatment (AFM1 +OTA). The study period lasted 28 days. The jejunum tissue was collected for the histological assessment and whole transcriptome analysis, and the whole blood was collected, and determination of serum biochemical indicators. The phenotypic results demonstrated that AFM1 and OTA caused intestinal barrier disruption via an increased apoptosis level and decreased expression of tight junction (TJ) proteins. The ceRNA network demonstrated that AFM1 and OTA induced cell apoptosis through activating the expression of DUSP9 and suppressing the expression of PLA2G2D, which were regulated by differentially expressed microRNAs (DEmiRNAs) (miR-124-y, miR-194-z, miR-224-x, and miR-452-x) and differentially expressed long non-coding RNAs (DElncRNAs) (FUT8 and GPR31C). And AFM1 and OTA decreased TJ proteins via inhibiting the expression of PAK6, which was regulated by several important DEmiRNAs and DElncRNAs. These DE RNAs in intestinal integrity were involved in MAPK and Ras signaling pathway. Overall, our findings expand the current knowledge regarding the potential mechanisms of intestinal integrity disruption brought on by AFM1 and OTA in vivo.

Detoxification of aflatoxin M1 in different milk types using probiotics.

The aim of this study, research the potential use of probiotics in reducing the toxic effect of Aflatoxin M1 in cow milk, goat milk, sheep milk, and Phosphate-buffered saline (PBS). Milk and Phosphate-buffered saline were contaminated with Aflatoxin M1 at a concentration of 100 ppt. Then, various study groups were formed by adding Lactobacillus acidophilus DSMZ 20079, Lactobacillus rhamnosus GG, and Bifidobacterium bifidum DSMZ 20456 probiotic bacteria at a density of 108 CFU/ml. Then, working groups were stored for 1 day and Aflatoxin M1 levels were analyzed by an Enzyme-Linked Immunosorbent Assay kit. The binding level of Aflatoxin M1 by probiotic bacteria varies between 2.32-12.52% in Phosphate-buffered saline, 9.08-40.14% in cow milk, 15.01-38.01% in goat milk, and 32.49-42.90% in sheep milk. The highest binding level of Aflatoxin M1 was detected in sheep milk and the lowest in Phosphate-buffered saline. The binding ability of Aflatoxin M1 is ranked from highest to lowest in sheep milk, cow milk, and goat milk. The data obtained from this study is important because it is the first study to show that if sheep and goat milk is enriched with probiotics, it can reduce AFM1 exposure.

Prevalence and concentration of Aflatoxin M1 in human breast milk in sub-Saharan Africa: a systematic review and meta-analysis, and cancer risk assessment.

This study aimed to assess the prevalence, concentration of AFM1 in human breast milk, and to determine the risk of cancer for infants in sub-Saharan Africa. A systematic literature search was performed using PubMed, CINAHL, Web of science, global health, Cochrane, and Google Scholar electronic databases. A random-effects model was used to estimate the pooled prevalence and concentration of AFM1 in breast milk. The meta-analysis of 8 articles containing 9 studies showed the pooled prevalence of AFM1 in breast milk to be 56.18% (95% CI: 29.65-82.71) and the pooled concentration to be 31.12 ng/L (95% CI: 25.97-36.25). The cancer risk assessment indicated for both male and female 1-month infants in Sierra Leone (HI > 1) is high, and all the rest of the infants are free of risk (HI < 1). The pooled prevalence and mean concentration of AFM1 in breast milk is high. Monitoring of AFB1 concentration of commonly used foods will be of high value in reducing the burden of AFM1.

The occurrence of aflatoxin M1 in milk samples of Iran: a systematic review and meta-analysis.

In this study, the average level of aflatoxin M1 in various types of milk from 107 articles (297 studies with 16,274 milk samples) were meta-analyzed using random-effect model based on the milk varieties (animal species and heating processes), geographical regions, seasons, detection techniques and dairy farming subgroups. Studies on milk contamination with aflatoxin M1 in Iran were collected using universal and Persian databanks from January 1974 to the end of November 2021. The overall aflatoxin M1 mean concentration and prevalence in milk samples of Iran were 39.65 ng/l (95% CI: 36.00-43.30) and 80% (95% CI: 76-85%), respectively. The rank order of importance of various variables in mean levels of aflatoxin M1 in milk samples included milk type (animal species) > geographical regions > detection techniques > dairy farming types > milk types (heating processes) > seasons. Findings revealed that the overall content of aflatoxin M1 in milk samples of Iran was lower than that allowed by the European Union, Institute of Standards and Industrial Research of Iran, and the USA, possibly due to the milk monitoring by the Iranian regulatory systems.

Hue Recognition Competitive Fluorescent Lateral Flow Immunoassay for Aflatoxin M1 Detection with Improved Visual and Quantitative Performance.

Immunological detection of small molecules in a point-of-care (POC) format is of great significance yet remains challenging for accurate visual discrimination and quantitative analysis. Here, we report a novel hue recognition competitive fluorescent lateral flow immunoassay (HCLFIA) strip that allows both visual and quantitative detection of aflatoxin M1 (AFM1). The HCLFIA strip works on the basis of the ratiometric change of emission, arising from the overlap of fluorescence signals of two nanocomposites tagged with probe antibodies and coated antigens. A visually discernible orange-red-to-green fluorescence color change allows the naked eye semiquantitative readout of AFM1 around the threshold concentration (0.05 ng mL-1), yielding a visible detection limit of 0.02 ng mL-1. Moreover, using a custom smartphone-based device and color chart analysis, ultrasensitive quantitative detection of AFM1 can be achieved with a low limit of detection at 0.0012 ng mL-1, which is considerably better than those of the previously reported colorimetric and fluorescent strips. The accuracy performed in spiked milk samples ranged from 97.91 to 113.12% with a coefficient of variation below 7.8%, showing good consistency with the results from isotope dilution liquid chromatography-tandem mass spectrometry. Thanks to the unique hue recognition scheme, the HCLFIA strip holds great potential for POC detection of small molecules.

Aflatoxin exposure among children of age 12-59 Months in Butajira District, South-Central Ethiopia: a community based cross-sectional study.

BACKGROUND: The continued provision of safe food, free of aflatoxin remains a huge challenge in developing countries. Despite several favourable climatic conditions that facilitate aflatoxin contamination in Ethiopia, there is little information showing aflatoxin exposure in children. Therefore, this study assessed aflatoxin exposure among young children in Butajira district, South-Central Ethiopia. METHODS: Community based cross-sectional study stratified by agro-ecology was employed in Health and Demographic Surveillance Site (HDSS) of Butajira. The study included 332 children aged 12-59 months and were selected by simple random sampling technique using the HDSS registration number as a sampling frame. We collected data on dietary practice and aflatoxin exposure. Aflatoxin M1 concentration in urine was measured by Enzyme-Linked Immunosorbent assay (ELISA). The data analysis was carried out using STATA. RESULTS: Detectable urinary Aflatoxin M1 was found in 62.4% (95% CI: 56.9 - 67.5%) of the children at a level ranging from 0.15 to 0.4 ng/ml. Children living in lowland agro-ecological zone had [AOR = 2.11 (95% CI; 1.15, 3.88] odds of being exposed to aflatoxin as compared to children living in highland agro-ecological zone. Children at lower socio-economic status [AOR = 0.27 (95% CI; 0.14, 0.50] and medium socio-economic status [AOR = 0.47 (95% CI; 0.25, 0.87] had 73% and 53% lower odds of being exposed to aflatoxin as compared to children in the higher socio-economic status, respectively. CONCLUSIONS: Aflatoxin exposure among young children was very high in South-Central Ethiopia. This high aflatoxin exposure might emphasize the need for aflatoxin exposure mitigation strategies in Ethiopia. Especially, raising awareness of the community towards aflatoxin exposure is very crucial. In addition, further research is required to assess long-term aflatoxin exposure and its association with child growth and development.

A novel electrochemical aptasensor based on layer-by-layer assembly of DNA-Au@Ag conjugates for rapid detection of aflatoxin M1 in milk samples.

Aflatoxin M1 (AFM1) is a common toxin in dairy products that causes acute and chronic human health disorders. Thus, the development of a rapid and accurate AFM1 detection method is of vital importance for food safety monitoring. This work was to develop a novel electrochemical aptasensor for sensitive and specific determination of AFM1. The dendritic-like nanostructure was formed on the gold electrode surface by layer-by-layer assembly of gold-silver core-shell nanoparticles modified with DNA conjugates. In the presence of AFM1, the specific recognition between AFM1 and Apt caused the disassociation of the DNA controlled dual Au@Ag conjugates from the surface of the electrode, causing less methylene blue to bind to the surface and weakening the electrochemical signal. The more AFM1 there is, the weaker the electrochemical signal. Transmission electron microscope results showed that the successfully synthesized Au@Ag nanoparticles exhibited a core-shell structure with Au as core and Ag as shell, and their average diameter was about 30 nm. Under optimal conditions, the electrochemical aptasensor showed a wide detection ranging from 0.05 ng mL-1 to 200 ng mL-1, and a low detection limit of 0.02 ng mL-1. Moreover, the proposed strategy has been successfully applied to the detection of AFM1 in cow, goat, and sheep milk samples with satisfactory recoveries ranging from 91.10% to 104.05%. This work can provide a novel rapid detection method for AFM1, and also provide a new sensing platform for the detection of other toxins.

A Strategy for Sample Preparation: Using Egg White Gel to Promote the Determination of Aflatoxin M1 Content in Milk Samples.

The analysis of food samples is a challenging task. The high complexity of food matrices hinders the extraction and detection of analytes from them. Therefore, the correct preparation of food samples is a crucial step for their subsequent analysis, as it achieves the proper isolation and preconcentration of analytes and removes the interfering proportion of the food matrix before instrumental analysis. We aimed to develop a method that not only satisfies the requirement of detecting trace compounds in complex matrices but also achieves a "greener" approach by reducing the use of organic solvents and non-degradable materials to minimize the health hazards posed to the operators as well as pollution to the environment. In this study, we prepared egg white as a concentrated gel and used this material for the biological purification of milk samples. After the milk protein was removed by acidification and salting, the residual amount of aflatoxin M1 in milk samples was quantitatively determined by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the novel egg white purification method possessed advantages over the immunoaffinity technique used as the reference method in extraction recovery, sensitivity, repeatability, and operability. The limit of detection (LOD) was 0.001 µg/kg. In spiked samples containing 0.01 µg/kg to 2 µg/kg of AFM1, the average recovery was 88.3-94.7%, with a precision of 6.1-11.0%. Improved repeatability was obtained by significantly reducing the operation time and resource requirements compared with the immunoaffinity technique currently used internationally. This study provides a reference for the further improvement of the relevant international standards in place for the detection of aflatoxin M1 in milk.