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.

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.

Prediction and validation of enzymatic degradation of aflatoxin M1: Genomics and proteomics analysis of Bacillus pumilus E-1-1-1 enzymes.

Aflatoxins are a class of highly toxic mycotoxins. Aflatoxin M1 (AFM1) is hydroxylated metabolite of aflatoxin B1, having comparable toxicity, which is more commonly found in milk. In this study, the whole genome sequencing of Bacillus pumilus E-1-1-1 isolated from feces of 38 kinds of animals, having aflatoxin M1 degradation ability was conducted. Bacterial genome sequencing indicated that a total of 3445 sequences were finally annotated on 23 different cluster of orthologous groups (COG) categories. Then, the potential AFM1 degradation proteins were verified by proteomics; the properties of these proteins were further explored, including protein molecular weight, hydrophobicity, secondary structure prediction, and three-dimensional structures. Bacterial genome sequencing combined with proteomics showed that eight genes were the most capable of degrading AFM1 including three catalases, one superoxide dismutase, and four peroxidases to clone. These eight genes with AFM1 degrading capacity were successfully expressed. These results indicated that AFM1 can be degraded by Bacillus pumilus E-1-1-1 protein and the most degrading proteins were oxidoreductases.

Degradation products of aflatoxin M1 (AFM1) formed by high voltage atmospheric cold plasma (HVACP) treatment.

Cold plasma technology is a novel non-thermal technology that has shown promising results for food decontamination and improving food safety. This study is a continuation of a previous investigation of the treatment of AFM1-contaminated skim and whole milk samples by HVACP. Previous research has shown HVACP is effective in degrading aflatoxin M1 (AFM1) in milk. The goal of this study is to identify the degradation products of AFM1 after HVACP treatment in pure water. An HVACP direct treatment at 90 kV using modified air (MA65: 65% O2, 30% CO2, 5% N2) was performed for up to 5 min at room temperature on a 5.0 mL water sample in a Petri dish artificially contaminated with 2 µg/mL of AFM1. The degradants of AFM1 were analyzed and their molecular formulae were elucidated by using high-performance liquid-chromatography time-of-flight mass spectrometry (HPLC-TOF-MS). Three main degradation products were observed and based on mass spectrometric fragmentation pathways, chemical structures for the degradation products were tentatively assigned. According to the structure-bioactivity relationship of AFM1, the bioactivity of the AFM1 samples treated with HVACP was reduced due to the disappearance of the C8-C9 double bond in the furofuran ring in all of the degradation products.

Three recombinant peroxidases as a degradation agent of aflatoxin M1 applied in milk and beer.

The aim of this work was to estimate the effects of three recombinant peroxidases (rPODs) on the degradation of aflatoxin M1 (AFM1) in a model solution and were applied in milk and beer to study the AFM1 degradation. Besides, the contents of AFM1 in model solution, milk and beer were evaluated, and the kinetic parameters of rPODs were determined (Michaelis-Menten constant - Km and maximal velocity - Vmax). The optimized reaction conditions (The degradation was over 60 %) for these three rPODs in the model solution were, respectively as follows: pH were 9, 9, and 10; hydrogen peroxide concentrations were 60, 50, and 60 mmol/L; at an ionic strength of 75 mmol/L and reaction temperature of 30 °C with 1 mmol/L K+ or 1 mmol/L Na+. These three rPODs (1 U /mL) presented the maximum activity for degradation of AFM1 of 22.4 %, 25.6 %, and 24.3 % in milk, while 14.5 %, 16.9 %, and 18.2 % in beer, respectively. Meanwhile, the survival rate of Hep-G2 cells raised about 1.4 times after treated with peroxidase-generated AFM1 degradation products. Therefore, POD may be a promising alternative to reduce the pollution of AFM1 in model solution, milk, beer, and minimize their impact on the environment in humans.

Efficacy of Bentonite and Fumonisin Esterase in Mitigating the Effects of Aflatoxins and Fumonisins in Two Kenyan Cattle Breeds.

The objective of the study was to investigate the efficacy of bentonite and fumonisin esterase, separately or combined, in mitigating the effects of aflatoxins (AF) and fumonisins (FUM) in Boran and Friesian-Boran crossbreed cattle. These effects were studied by measuring mycotoxins, their metabolites, and biomarkers that relate to animal health, productivity, and food safety. The study was divided into three experiments each lasting for 2 weeks. Cows in experiment 1 received in random order aflatoxin B1 (AFB1) [788 µg/cow/day (69.7 µg/kg dry matter intake (DMI)) for Borans and 2,310 µg/cow/day (154 µg/kg DMI) for crossbreeds], bentonite (60 g/cow/day), or both AFB1 and bentonite. Boran cows in experiment 2 received in random order FUM (12.4 mg/cow/day (1.1 mg/kg DMI)), fumonisin esterase (120 U/cow/day), or both FUM and fumonisin esterase. Boran cows in experiment 3 received in random order AFB1 (952 µg/cow/day (84.2 µg/kg DMI)) + FUM (30.4 mg/cow/day (2.7 mg/kg DMI)), bentonite (60 g/cow/day) + fumonisin esterase (120 U/cow/day), or both AFB1 + FUM and bentonite + fumonisin esterase. Feeding AFB1 and/or FUM contaminated feed with or without the addition of the detoxifiers for 14 days did not affect DMI, milk composition, hematology, and blood biochemical parameters. The addition of bentonite in a diet contaminated with AFB1 led to a decrease in milk aflatoxin M1 (AFM1) concentration of 30% and 43%, with the carry-over subsequently decreasing from 0.35% to 0.20% and 0.08% to 0.06% for crosses and Borans, respectively. No significant change was observed in the sphinganine/sphingosine (Sa/So) ratio following feeding with FUM alone or in combination with fumonisin esterase; however, the ability of fumonisin esterase to hydrolyze FUM into less toxic fully hydrolyzed FUM and partially hydrolyzed FUM was evident in the rumen fluid and feces. These results indicate bentonite was effective in decreasing AFM1 concentration in milk, and AFB1 and AFM1 in plasma, while fumonisin esterase can convert FUM into less toxic metabolites and can be a suitable addition to feed cocontaminated with AFB1 and FUM.

Association between aflatoxin M1 excretion in milk and indicators of rumen fermentation in bovines.

Aflatoxins and its metabolites negatively impact the ruminant health and production. The present cross-sectional study was aimed to determine the effect of aflatoxins on rumen fermentation by deducing the correlation between the aflatoxin M1 (AFM1) excretion in milk and indicators of rumen fermentation in bovines. The indicators of rumen fermentation were taken into account and correlated with AFM1 concentration in milk of 120 bovines (cattle (n = 82) and buffalo (n = 38)). The AFM1 in milk samples (n = 120) was quantified by ELISA kit. The correlation analysis revealed that with increase in excretion of AFM1 in milk, the pH (r = 0.38), methylene blue reduction time (MBRT) (r = 0.43), sedimentation activity time (SAT) (r = 0.31) and ammonia nitrogen content (r = 0.34) of rumen liquor increase, whereas the total volatile fatty acid (TVFA) content (r = - 0.25), total bacterial count (TBC) (r = - 0.43) and total protozoal count (TPC) (r = - 0.14) of rumen liquor decrease. The results of the present study suggest that the presence of aflatoxins in rumen could have negative effect on the process of rumen fermentation. Therefore, the prevention of primary entry point(s) of AFB1 through the feed of bovines is important for the animal health as well as public health.

Metabolomic Analysis Reveals the Mechanisms of Hepatotoxicity Induced by Aflatoxin M1 and Ochratoxin A.

Aflatoxin M1 (AFM1) is the only toxin with the maximum residue limit in milk, and ochratoxin A (OTA) represents a common toxin in cereals foods. It is common to find the co-occurrence of these two toxins in the environment. However, the interactive effect of these toxins on hepatoxicity and underlying mechanisms is still unclear. The liver and serum metabolomics in mice exposed to individual AFM1 at 3.5 mg/kg b.w., OTA at 3.5 mg/kg b.w., and their combination for 35 days were conducted based on the UPLC-MS method in the present study. Subsequent metabolome on human hepatocellular liver carcinoma (Hep G2) cells was conducted to narrow down the key metabolites. The phenotypic results on liver weight and serum indicators, such as total bilirubin and glutamyltransferase, showed that the combined toxins had more serious adverse effects than an individual one, indicating that the combined AFM1 and OTA displayed synergistic effects on liver damage. Through the metabolic analysis in liver and serum, we found that (i) a synergistic effect was exerted in the combined toxins, because the number of differentially expressed metabolites on combination treatment was higher than the individual toxins, (ii) OTA played a dominant role in the hepatoxicity induced by the combination of AFM1, and OTA and (iii) lysophosphatidylcholines (LysoPCs), more especially, LysoPC (16:1), were identified as the metabolites most affected by AFM1 and OTA. These findings provided a new insight for identifying the potential biomarkers for the hepatoxicity of AFM1 and OTA.

The efficacy of clay bentonite, date pit, and chitosan nanoparticles in the detoxification of aflatoxin M1 and ochratoxin A from milk.

Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are highly toxic mycotoxin metabolites that are found as food pollutants, posing health risks to humans and animals. The objective of the current study is to establish a sensitive, reliable method for determining AFM1 and OTA using high-performance liquid chromatography (HPLC) and attempting to assess the efficacy of bentonite, date pit, and chitosan nanoparticles for AFM1 and OTA detoxification from contaminated milk. As revealed, AFM1 was found in 65.7% of analyzed samples ranging from 4.5 to 502 ng/L, while 25.7% of examined samples contained OTA ranging from 1.45 to 301 ng/L. Furthermore, for AFM1 and OTA. The advanced procedure was thoroughly validated by evaluating linearity (R2 > 0.999), LOD (0.9615 and 0.654 ng/L), and LOQ (2.8846 and 1.963 ng/L), recovery (93-95% and 87-91%), as well as precision (≤ 1%RSD). The experimental data revealed a higher removal efficiency of bentonite and date pit than chitosan nanoparticles in the case of AFM1 (68%, 56%, and 12%) and OTA (64%, 52%, and 10%), respectively with slight change in nutritional milk components like fat, protein, and lactose. Eventually, it is concluded that bentonite and date pit can be considered efficient adsorbing agents to extract AFM1 and OTA from contaminated milk.

Aflatoxin B1 and Aflatoxin M1 Induce Compromised Intestinal Integrity through Clathrin-Mediated Endocytosis.

With the growing diversity and complexity of diet, humans are at risk of simultaneous exposure to aflatoxin B1 (AFB1) and aflatoxin M1 (AFM1), which are well-known contaminants in dairy and other agricultural products worldwide. The intestine represents the first barrier against external contaminants; however, evidence about the combined effect of AFB1 and AFM1 on intestinal integrity is lacking. In vivo, the serum biochemical parameters related to intestinal barrier function, ratio of villus height/crypt depth, and distribution pattern of claudin-1 and zonula occluden-1 were significantly affected in mice exposed to 0.3 mg/kg b.w. AFB1 and 3.0 mg/kg b.w. AFM1. In vitro results on differentiated Caco-2 cells showed that individual and combined AFB1 (0.5 and 4 µg/mL) and AFM1 (0.5 and 4 µg/mL) decreased cell viability and trans-epithelial electrical resistance values as well as increased paracellular permeability of fluorescein isothiocyanate-dextran in a dose-dependent manner. Furthermore, AFM1 aggravated AFB1-induced compromised intestinal barrier, as demonstrated by the down-regulation of tight junction proteins and their redistribution, particularly internalization. Adding the inhibitor chlorpromazine illustrated that clathrin-mediated endocytosis partially contributed to the compromised intestinal integrity. Synergistic and additive effects were the predominant interactions, suggesting that these toxins are likely to have negative effects on human health.

Field Monitoring of Aflatoxins in Feed and Milk of High-Yielding Dairy Cows under Two Feeding Systems.

Aflatoxin M1 (AFM1) is a hydroxylated metabolite of aflatoxin B1 (AFB1) that can be excreted in milk of cows after consuming contaminated feed. The aim of this study consisted of a field monitoring to assess the contamination levels of AFB1 in 60 feed samples from two feeding systems for high-yielding dairy cows and of AFM1 in the corresponding raw milk samples. The aflatoxins were analyzed by in-house validated methods based on high-performance liquid chromatography (HPLC) with fluorescence detection. AFB1 was detected in 55% of feed samples (mean 0.61 µg/kg, with 2 samples exceeding the European Union (EU) maximum level set at 5 µg/kg), with greater incidence and concentration in compound feed than in unifeed rations (p < 0.05). AFM1 was detected in 38.3% milk samples (mean 12.6 ng/kg, with 5 samples exceeding the EU maximum level set at 50 ng/kg), with a higher occurrence in milk of cows fed compound feed, as well as in spring milk compared to that produced in winter. The overall transfer ratio of aflatoxins from feed to milk was 3.22%, being higher in cows fed with compound feed and in spring milkings. In a selection of positive matched samples (n = 22), the ratio AFM1/AFB1 exceeded the European Food Safety Authority (EFSA) estimated 6% threshold for high-yielding dairy cows.

Immunological effects of AFM1 in experimental subchronic dosing in mice prevented by lactic acid bacteria.

AIM: Recently, higher contamination by aflatoxin M1 (AFM1) has been detected in many countries. Unfortunately, many tons of contaminated milk and milk byproducts are removed from the food chain to avoid human contamination; as a consequence of higher economic losses. Fewest number of studies are interested to AFM1 detoxification using lactic acid bacteria. MATERIALS AND METHODS: In this study, AFM1-degradation using Lactobacillus paracasei BEJ01 (LPBEJ01) was tested in vitro. The preventive effect of LPBEJ01 against AFM1 immunobiological effects in mice are treated orally during 3 weeks with 100 µg AFM1, LPBEJ01 (2 × 109 CFU/ml∼2 mg/kg p.c.) and a mixture of AFM1 and LPBEJ01. RESULTS: In vitro LPBEJ01 was found able to absorb 98% of AFM1 (100 µg/ml) in liquid medium after 24 h and more than 95% of AFM1 could be eliminated after 24 h in a solid-state fermentation. Animals treated with AFM1 obtained lower body weight than the control ones. The mitogenic response of spleen mononuclear cells (SMCs) in vivo was higher in mice treated with AFM1. The SMC of mice treated with AFM1 produced lower levels of IL-2, higher levels IL-4 and no effect on IL-10 production. The peritoneal macrophages of mice that treated with AFM1 released less H2O2, while mice exposed orally with the mixture of AFM1 and LPBEJ01 produced higher levels. CONCLUSION: LPBEJ01 was safe and it did not have any sign of toxicity. It can be used as an additive for AFM1-detoxification contamination in the food chain in countries suffering from this problem.

Aflatoxin M1 prevalence in breast milk in Morocco: Associated factors and health risk assessment of newborns "CONTAMILK study".

The aim of this study was to assess the contamination of breast milk by aflatoxin M1 among nursing mothers from Rabat, Morocco, and to explore its association with several maternal parameters and dietary habits. In addition, the health risk assessment of the newborns by the estimation of the daily intake. A competitive Enzyme Linked Immunosorbent Assay method was used for the analysis of aflatoxin M1 in breast milk samples. Analytical results indicate that out of 82 total samples, 43 samples (52.4%) of milk were positive. Aflatoxin M1 levels ranged from undetectable to 13.33 ng/L, while the mean level was 5.75 ± 3.44 ng/L. Besides, several factors and foodstuffs seem to increase the level of AFM1 in breast milk. As regards the estimated daily intake of aflatoxin M1, it varies between immeasurable and a maximum of 1.16 ng/kg.bw. The degree of exposure to AFB1 and the levels of its metabolite AFM1 in breast milk were low, compared to some studies from other countries. Further investigations and periodic monitoring programs are recommended in large samples and in many cities of morocco to assess the level of exposure of the Moroccan population.

An assessment of the occurrence and nutritional factors associated with aflatoxin M1, ochratoxin A, and zearalenone in the breast milk of nursing mothers in Hamadan, Iran.

This research investigated the occurrence of aflatoxin M1 (AFM1), ochratoxin A (OTA), and zearalenone (ZEN) in human milk samples in the Hamadan city, Iran. The study was carried out using the milk of nursing mothers from ten governmental health care centers. Mycotoxin content of ninety milk samples measured using enzyme-linked immunosorbent assay (ELISA). All samples that tested positive for AFM1 with the ELISA test were subsequently analyzed by high-performance liquid chromatography (HPLC). The mean ± SD concentrations of AFM1, determined by ELISA and HPLC were 5.98 ± 1.47 and 4.36 ± 1.23 ng/L, respectively. OTA and ZEN levels were below the detection limit (<5 ng/L) in all samples. None of the contaminated samples exceeded the regulation limit set by the European Commission (25 ng/L) for AFM1 in infant formula. We found a significant correlation between the AFM1 concentration in breast milk and infant age and milk consumption by the nursing mother (p < 0.05). These findings revealed that infants are susceptible to AFM1 exposure from their mother's milk. The authors recommend that additional research be conducted on the analysis of foodstuff and biological fluids for various mycotoxins.

Aflatoxin M1-Binding Ability of Selected Lactic Acid Bacteria Strains and Saccharomyces boulardii in the Experimentally Contaminated Milk Treated with Some Biophysical Factors.

There is a growing concern regarding the recurrent observation of aflatoxins (AFs) in the milk of lactating animals. Regarding this, the present study was conducted to assess the aflatoxin M1 (AFM1)-binding ability of three species, namely Lactobacillus rhamnosus, L. plantarum, and Saccharomyces boulardii, inAFM1-contaminatedmilk. The mentioned species were administeredatthe concentrations of107 and 109 CFU/mLto skimmed milk contaminated with 0.5 and 0.75 ng/mL AFM1 within the incubation times of 30 and 90 min at 4&deg;C and 37&deg;C. Lactobacillus rhamnosus was found to have the best binding ability at the concentrations of 107 and 109 (CFU/ml), rendering 82% and 90% removal in the milk samples with 0.5 and 0.75 ng/ml AFM1, respectively. Accordingly, this value at 107 and 109 CFU/ml of L. plantarum was obtained 89% and 82% with 0.75 ng/ml of AFM1, respectively. For S. boulardii at 107 and 109 CFU/ml, the rates were respectively estimated at 75% and 90% with 0.75 ng/ml of AFM1. The best AFM1-binding levels for L. rhamnosus, L. plantarum, and S. boulardii were 91.82&plusmn;10.9%, 89.33&plusmn;0.58%, and 93.20&plusmn;10.9, respectively, at the concentrations of 1&times;109, 1&times;107, and 1&times;107 CFU/ml at 37, 4, and 37&deg;C, respectively. In this study, the maximum AFM1 binding (100.0&plusmn;0.58) occurred while a combination of the aforementioned probiotics was employed at a concentration of 1&times;107 CFU/ml at 37&deg;C with 0.5 ng/ml AFM1, followed by the combination of L. rhamnosus and L. plantarum (95.86&plusmn;10.9) at a concentration of 1&times;109 CFU/ml at the same temperature with 0.75 ng/ml AFM1. It was concluded that the use of S. boulardii in combination with Lactobacillus rhamnosus and L. plantarum, which bind AFM1 in milk, can decrease the risk of AFM1 in dairy products.

Transcriptomic and proteomic profiling reveals the intestinal immunotoxicity induced by aflatoxin M1 and ochratoxin A.

Mycotoxins-contaminated milk could threaten human health; therefore, it is necessary to demonstrate the toxicological effect of mycotoxins in milk. Most recently, researchers have paid more attention to the immunotoxic effects of the individual cereal-contaminating mycotoxins, namely, zearalenone and deoxynivalenol. However, there is scant information about the intestinal immunotoxicity of aflatoxin M1 (AFM1), let alone that of a combination of AFM1 and ochratoxin A (OTA), which often co-occur in milk. To reveal the inflammatory response caused by these mycotoxins, expression of inflammation-related genes in differentiated Caco-2 cells was analyzed, demonstrating a synergistic effect of the mixture of AFM1 (4 µg/mL) and OTA (4 µg/mL). Integrative transcriptomic and proteomic analyses were also performed. A cross-omics analysis identified several mechanisms underlying this synergy: (i) compared with stimulation with either compound alone, combined use resulted in stronger induction of proteins involved in immunity-related pathways; (ii) combination of the two agents targeted different points in the same pathways; and (iii) combination of the two agents activated specific inflammation-related pathways. These results suggested that combined use of AFM1 and OTA might exacerbate intestinal inflammation, indicating that regulatory authorities should pay more attention to food contamination by multiple mycotoxins when performing risk assessments.

Control of aflatoxin M1 in milk by novel methods: A review.

Aflatoxin M1 (AFM1) in milk and milk products has been recognised as an issue for over 30 years. Controlling AFM1 in milk is important to protect human health and trade. Preventing contamination by avoiding fungal contamination of cattle feed is the best method of control, however this is hard to avoid in some countries. Treating milk containing AFM1 is an alternative control measure, however, there is no single approved method. The challenge is to select a treatment method that is effective but does not affect the organoleptic quality of milk. This study reviews the strategies for degrading AFM1 in milk including yeast, lactic acid bacteria, enzyme, peroxide, ozone, UV light and cold plasma. This review compares the efficacy, influencing factors, (possible) mechanisms of activity, advantages, limitations and potential future trends of these methods and provides some recommendations for the treatment of milk to reduce the risk of AFM1 contamination.

Probiotic bacteria and yeasts adsorb aflatoxin M1 in milk and degrade it to less toxic AFM1-metabolites.

The presence of contaminants such as aflatoxins (AFs) in dairy products constitutes a serious risk to the health of consumers, especially children who are most sensitive to the adverse effects of AFs. The presence of Aflatoxin M1 (AFM1) in milk is a public health problem since dairy products are massively consumed worldwide. The aim of the present work was to select microorganisms capable of reducing AFM1 entry into the food chain through adsorption/degradation strategies. Moreover, the toxicity of AFM1 degradation products was evaluated. All tested strains had the capacity to adsorb 19%-61% AFM1 in milk. These strains also had the ability to degrade AFM1 into metabolites less toxic than the original toxin. Moreover, this is the first study to report harmless and probiotic Pediococcus pentosaceus and Kluveromyces marxianus have the ability to adsorb and degrade AFM1 to less toxic metabolites in milk.

Purification, characterization, and functional groups of an extracellular aflatoxin M1 -detoxifizyme from Bacillus pumilus E-1-1-1.

The experiment was conducted to purify high activity extracellular enzymes, which were produced by a strain that we previously screened was able to degrade aflatoxin effectively, and speculate the functional groups of the enzyme associated with degradation. An extracellular aflatoxin-detoxifizyme (DAFE) from Bacillus pumilus E-1-1-1 was purified through a process including ammonium sulfate precipitation, ultrafiltration, Sephadex chromatography, and ion exchange chromatography. The molecular mass of the enzyme assessed by SDS-PAGE was found to be approximately 58 kDa. The optimum reaction temperature and pH for the purified enzyme were 45°C and pH 7, respectively. The enzyme showed temperature stability of up to 60°C. Ba2+ , Ca2+ Na+ , Mn2+ , EDTA, and ß-mercaptoethanol showed inhibitory effects on the enzyme activity. Mg2+ , Fe3+ , Zn2+ and K+ were the activators of enzymes. This enzyme was composed of at least 15 kinds of amino acids. Lysine, tryptophan, and histidine residues were necessary and major functional groups to maintain enzyme activity, disulfide bonds were observed, serine residues had little effect on the enzyme activity, so it was not the necessary group to reflect the enzyme activity, and arginine had no effect on enzyme activity.

Surface screening, molecular modeling and in vitro studies on the interactions of aflatoxin M1 and human enzymes acetyl- and butyrylcholinesterase.

Aflatoxin M1 (AFM1) is a mycotoxin produced by Aspergillus fungi and found in contaminated milk, breastfeed and dairy products, being highly toxic and carcinogenic to humans and other mammalian species. It is also produced in the human body as a metabolite of aflatoxin B1 (AFB1), one of the most toxic natural products known. Previous studies have shown that AFM1 is a potential inhibitor of the enzyme acetylcholinesterase (AChE), and therefore, a potential neurotoxic agent. In this work, surface screening (SS) and molecular dynamics (MD) simulation on human acetylcholinesterase AChE (HssAChE) were performed to corroborate literature data regarding preferential binding sites and type of inhibition. Also, an inedited theoretical study on the interactions of AFM1 with human butyrylcholinesterase (HssBChE) was performed. In vitro inhibition tests on both enzymes were done to support theoretical results. MD simulations suggested the catalytic anionic site of HssAChE as the preferential binding site for AFM1 and also that this metabolite is not a good inhibitor of HssBChE, corroborating previous studies. In vitro assays also corroborated molecular modeling studies by showing that AFM1 did not inhibit BChE and was able to inhibit AChE, although not as much as AFB1.