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.

Biodiversity and biotechnological properties of lactic acid bacteria isolated from traditional Moroccan sourdoughs.

The present study aimed at characterizing lactic acid bacteria (LAB) strains isolated from traditional sourdoughs collected in different regions of Morocco. Isolated strains were firstly identified using Gram staining and catalase reaction test. Presumptive LAB strains were then checked for various phenotypical properties including growth at 45 °C, resistance to NaCl, enzyme production, acidification capacity, diacetyl and exopolysaccharide (EPS) production, and antifungal activity. Finally, selected LAB strains were identified using 16S rDNA sequencing. Results showed that 32.1% of the isolates were thermophilic (45 °C) and 83.9% were resistant to NaCl (6.5%). Moreover, 51.7 and 37.5% were able to produce diacetyl and EPS, respectively. Regarding enzyme production, 55.3 and 7.1% of the isolates showed lipolytic and proteolytic activities, respectively. Low pH values (3.37-3.76) were obtained after 24 h of incubation of LAB strains in de Man, Rogosa and Sharpe (MRS) broth. Antifungal activity test against Aspergillus flavus, Aspergillus niger and Penicillium spp. showed an inhibition rate up to 50%. Bacterial DNA sequencing showed that LAB isolates belong to seven species, chiefly Levilactobacillus brevis, Lentilactobacillus parabuchneri, Lactiplantibacillus plantarum, Pediococcus pentosaceus, Enterococcus hirae, Bifidobacterium pseudocatenulatum, and Companilactobacillus paralimentarius. These findings, for the first time in Moroccan sourdoughs, indicate that the isolated LAB strains have good multifunctional properties and could be suitable as good starters for sourdough bread production under controlled conditions.

Prevention and Detoxification of Mycotoxins in Human Food and Animal Feed using Bio-resources from South Mediterranean Countries: a Critical Review.

Mycotoxins, which are natural toxic compounds produced by filamentous fungi, are considered major contaminants in the food and feed chain due to their stability during processing. Their impacts in food and feedstuff pollution were accentuated due the climate change in the region. They are characterized by their toxicological effects on human and animal health but also by their harmful economic impact. Mediterranean countries: Algeria, Egypt, Libya, Morocco and Tunisia are characterized by high temperatures and high relative humidity, particularly in littoral regions that provide favorable conditions for fungal growth and toxinogenesis. Many scientific papers have been published recently in these countries showing mycotoxin occurrence in different commodities and an attempt at bio-detoxification using many bio-products. In order to minimize the bioavailability and/or to detoxify mycotoxins into less toxic metabolites (bio-transforming agents), safe and biological methods have been developed including the use of lactic acid bacteria, yeasts, plant extracts and clays minerals from Mediterranean regions. The aim of this review is to present the pollution of mycotoxins in food and feedstuff of humans and animals and to discuss the development of effective biological control for mycotoxin removal/detoxification and prevention using bio-products. This review will also elucidate the new used natural products to be considered as a new candidates for mycotoxins detoxification/prevention on animal feedstuffs.

Occurrence of pre- and postharvest multi-mycotoxins in durum wheat grains collected in 2020 and 2021 in two climatic regions of Tunisia.

Twenty two mycotoxins in 136 durum wheat collected from Tunisia in 2020 and 2021 were investigated. Mycotoxins were analyzed by UHPLCMS/MS. In 2020, 60.9% of the samples were contaminated with Aflatoxin B1 (AFB1) and/or enniatin. Whereas, in 2021, 34.4% were contaminated by enniatins. AFB1 was detected only in 2020, in the continental region (6/46) and all samples exceeded limits. AFB1 was detected in stored wheat (24-37.8 µg/kg) but also in pre-stored wheat (17-28.4 µg/kg) and in one sample collected in the field (21 µg/kg). Enniatin A1, enniatin B and enniatin B1 were detected in wheat collected in the field (30-7684 µg/kg), pre-storage (42-1266 µg/kg) and storage (65.8-498.2 µg/kg) from the continental region also, in sample collected in pre-storage (31.3-1410 µg/kg) and at harvest (48- 1060 µg/kg). Samples had a water activity less than 0.7 and moisture content ranged between 09-14%. AFB1 level represent a health risk to the Tunisian consumers.

Visualisation and quantification of fumonisins bound by lactic acid bacteria isolates from traditional African maize-based fermented cereals, ogi and mahewu.

Consumption of fumonisin-contaminated foods has a negative influence on the health of humans (carcinogen; oesophageal cancer in Eastern Cape in South Africa). Lactic acid bacteria (LAB) have emerged as a promising natural detoxification agent against mycotoxins. The aim of this study was to visualise the interaction between fumonisins (FB1 and FB2) and LAB: Lactobacillus plantarum FS2, L. delbrueckii subsp. delbrueckii CIP 57.8T and Pediococcus pentosaceus D39, isolated from traditional fermented maize-based products (ogi and mahewu) using confocal laser scanning microscopy (CLSM) and to then quantify the LAB-bound fumonisin using high performance liquid chromatography (HPLC). The objective was to obtain a physically visible and quantifiable binding interaction between fumonisins and LAB strains with the aim of utilising LAB as a possible detoxifying agent. Fumonisins were derivatised using naphthalene-2,3-dicarboxaldehyde (NDA) and then combined with non-fluorescent LAB cells (viable and non-viable). For the quantification of bound fumonisins, viable and non-viable cells were incubated in the presence of predetermined concentrations of fumonisins and the level of fumonisin in the suspension was determined. CLSM showed the derivatised green fluorescent fumonisins binding to the surface of each of the LAB cells. For viable cells, L. plantarum FS2 bound FB1 most effectively while P. pentosaceus D39 bound the least level of FB1. The highest levels of FB2 were bound by L. plantarum R 1096 and the least by L. delbrueckii CIP 57.8 T. For non-viable cells, L. plantarum FS2 was also the most effective for binding both fumonisins with P. pentosaceus D39 and L. delbrueckii CIP 57.8 T being the least effective for FB1 and FB2, respectively. To our knowledge, this is the first study to visualise the interaction between LAB and fumonisins. We demonstrate that LAB isolates from indigenous fermented maize-based beverages bind fumonisins and thus present a potential strategy for their reduction in these traditional foods.

Development and in-house validation of a rapid and simple to use ELISA for the detection and measurement of the mycotoxin sterigmatocystin.

Sterigmatocystin (STG) is a highly toxic secondary fungal metabolite structurally closely related to the well-known carcinogenic aflatoxins. Its presence has been reported in grains and grain-based products as well as in other foodstuffs like nuts, green coffee beans, spices, beer and cheese. Due to the lack of suitable data on the occurrence of STG, in 2013, the European Food Safety Authority (EFSA) could not characterise its risk for human health and recommended that more data on STG in food and feed needed to be collected. In order to provide a new tool for the specific detection of STG, a competitive enzyme-linked immunosorbent assay (ELISA) was developed, optimised and validated in this study based on a sensitive monoclonal antibody specific to STG with no cross-reactivity with aflatoxins. The sample preparation method for rice, wheat and maize was based on a modified QuEChERS (quick, easy, cheap, effective, rugged and safe) approach. The assay was validated for the detection of STG in rice, wheat and maize in accordance with the guidelines for validation of semi-quantitative screening methods included in Commission Regulation (EU) 519/2014. The screening target concentration (STC) was set at 1.5 µg/kg. The cutoffs for rice, wheat and maize were 1.2, 1.2 and 1.3 µg/kg and the false suspected rates were 0.34, 1.15 and 0.78%, respectively. Good correlation was found between the results obtained by the STG ELISA and LC-MS/MS method for naturally contaminated rice samples. This validated method can be applied as a sensitive and high-throughput screening for the presence of STG in a range of agricultural commodities. Graphical abstract A new enzyme-linked immunosorbent assay based on an antibody specific to sterigmatocystin for the detection of this mycotoxin in corn, wheat and rice.

Aspergillus korhogoensis, a Novel Aflatoxin Producing Species from the Côte d'Ivoire.

Several strains of a new aflatoxigenic species of Aspergillus, A. korhogoensis, were isolated in the course of a screening study involving species from section Flavi found contaminating peanuts (Arachis hypogaea) and peanut paste in the Côte d'Ivoire. Based on examination of four isolates, this new species is described using a polyphasic approach. A concatenated alignment comprised of nine genes (ITS, benA, cmdA, mcm7, amdS, rpb1, preB, ppgA, and preA) was subjected to phylogenetic analysis, and resulted in all four strains being inferred as a distinct clade. Characterization of mating type for each strain revealed A. korhogoensis as a heterothallic species, since three isolates exhibited a singular MAT1-1 locus and one isolate exhibited a singular MAT1-2 locus. Morphological and physiological characterizations were also performed based on their growth on various types of media. Their respective extrolite profiles were characterized using LC/HRMS, and showed that this new species is capable of producing B- and G-aflatoxins, aspergillic acid, cyclopiazonic acid, aflavarins, and asparasones, as well as other metabolites. Altogether, our results confirm the monophyly of A. korhogoensis, and strengthen its position in the A. flavus clade, as the sister taxon of A. parvisclerotigenus.

Cooccurrence of mycotoxins in maize and poultry feeds from Brazil by liquid chromatography/tandem mass spectrometry.

The objective of this study was to quantitatively evaluate mycotoxins in samples of maize and poultry feed produced in Brazil. A multimycotoxin method based on HPLC-MS/MS was applied to investigate the occurrence of toxical fungal metabolites in 119 samples collected from poultry feed factory integrated poultry farms: maize grain (74), poultry feed (36), and feed factory residue (9). Twenty of 101 fungal metabolites investigated were detected and quantified in the samples: aflatoxins B1, B2, G1, and G2, fumonisins B1, B2, and B3, hydrolyzed fumonisin B1, zearalenone, agroclavine, chanoclavine, deoxynivalenol, and nivalenol, and enniatin A, A1, B, B1, beauvericin, kojic acid, and moniliformin. Most samples were contaminated with more than one mycotoxin. All samples were contaminated with fumonisins, with medians values of 1,840 µ g/kg, 239 µ g/kg, and 23,676 µ g/kg for maize, feed, and factory residue samples, respectively. Surprisingly, beauvericin was detected in more than 90% of samples. The median contaminations of aflatoxin and trichothecenes were low, near LOD values. The factory residue presented highest contamination levels for all mycotoxins. This is the first study dealing with agroclavine, chanoclavine, enniatin A, A1, B, B1, beauvericin, and kojic acid contamination of maize and poultry feeds from Brazil.