**1. Introduction**

Mycotoxins are secondary metabolites produced by microfungi that are capable of causing disease and death in humans and other animals [1]. The effects of some food-borne mycotoxins are acute with symptoms of severe illness appearing rapidly after consumption of food products contaminated with mycotoxins [2]. Of the several hundred mycotoxins identified so far, about a dozen have gained the most attention due to their severe effects on human health and their occurrences in food; and among the most commonly observed mycotoxins that present a concern to human health and livestock are aflatoxins [2].

Aflatoxins are amongst the most poisonous mycotoxins produced by species within *Aspergillus* section *Flavi*, which grow in soil, decaying vegetation, hay, grains, and various other substances [3,4]. Crops that are frequently affected by *Aspergillus* spp. include cereals (corn, sorghum, wheat, and rice), oilseeds (soybean, peanut, sunflower, and cotton seeds), spices (chili peppers, black pepper, coriander, turmeric, and ginger), and tree nuts (pistachio, almond, walnut, coconut, and Brazil nut) [1,5–9]. The four major aflatoxins are called aflatoxins B1, B2, G1, and G2 based on their fluorescence under UV light (blue or green). Among them, aflatoxin B1 (AFB1) is one of the most hazardous mycotoxins, primarily carcinogenic and genotoxic [10] and harmful to the liver [11]. The International Agency for Research on Cancer (IARC) classifies AFB1 as Group 1 carcinogen (carcinogenic to humans) [12]. In accordance with Regulation (EU) No 574/2011, the maximum permitted level for AFB1 in feed is 0.02 mg/kg.

Sterigmatocystin (ST) is a late metabolite in the aflatoxin pathway and is also produced as a final biosynthetic product by a number of species such as *Aspergillus versicolor* and *Aspergillus nidulans* [1,13]. ST is both mutagenic and tumorigenic but is less potent than aflatoxin [13]. Although experiments have shown genotoxicity and carcinogenicity of ST, limited data are available on the tumorigenic effect of the mycotoxin, which is why IARC classified it as a potential human carcinogen (Group 2B) in 1987 and has not revised this opinion ever since [14].

Lactic acid bacteria (LAB), Gram-positive, nonsporulating, oxidase and catalase negative, anaerobic aerotolerant microorganisms, are found in both the animal and the human body [15]. They ferment glucose to lactic acid. The most important genera are *Lactobacillus*, *Lactococcus*, *Leuconostoc*, *Enterococcus*, and *Pediococcus*, with 294, 21, 15, 59, and 11 species belonging to each genus, respectively [16].

Food-borne lactic acid bacteria able to bind mycotoxins can prevent their biotransformation to more toxic metabolites in the digestive tract, as the mycotoxin-microorganism adduct can pass through the body and be excreted in the feces, similarly as in the case of industrial mycotoxin binders, like aluminosilicates or glucomannan [17,18]. Numerous studies have shown that certain strains of some LAB species can bind mycotoxins, among them AFB1, to their surface [19–21]. The published results indicate that the adsorption of AFB1 to microorganisms is a rapid process. The binding involves the formation of a reversible complex between the chemically unmodified mycotoxin molecule and the microorganism surface, and the yield of AFB1 removal is dependent of the concentration of both the mycotoxin and the bacteria [22]. The binding mechanism is not yet elucidated, but the binding of AFB1 to the glycan components of the cell wall of probiotic bacteria has been suggested as a key momentum in the process [23–25].

According to literature data, the binding of aflatoxins by LAB strains is highly strain specific. The ratio of AFB1 bound by 10<sup>9</sup> cfu/mL of 8 strains of *L. casei* varied from 14% to 49% from the available 4.6 µg/mL in the studies of Hernandez-Mendoza et al. [26]. Reasons for the strain-specificity of AFB1 binding are yet unknown, but differences in cell wall components, particularly in the peptidoglycan content, may be implicated [27].

There has been quite a bit of research done on the ability of LAB to mitigate the detrimental effects of aflatoxin-producing fungal strains and their AFB1 binding capacity [20,22,28–30], though focusing mostly on lactobacilli and bifidobacteria. In addition, data on aflatoxin binding abilities of different strains belonging to the same species are scarce.

At our department, microbes with colony morphology of lactic acid bacteria were isolated on LAB selective MRS (de Man, Rogosa and Sharpe, VWR) plates from 14 exotic animals of the Budapest Zoo and Botanical Garden [31,32]. The identification of the strains was done by sequencing. At present, the collection comprises of nearly 1000 strains and is constantly expanding. Most of our strains belong to the genera *Lactobacillus* and *Enterococcus*, but we also managed to isolate strains belonging to the other LAB genera.

Our goal was to screen strains of the genus *Enterococcus*, *Lactococcus*, *Pediococcus*, and *Weissella* from our collection for AFB1 and ST binding capacities. In the literature, PBS medium (phosphate buffer solution) is most commonly used in mycotoxin binding assays of LAB strains. As our work was carried out as part of a probiotic development project, we chose a medium, MRS medium (Lactobacillus Agar according to DeMan, Rogosa and Sharpe), for our experiments, which is closer to

real conditions due to its much higher organic matter content and provides optimal conditions for the microbes. Mycotoxin concentrations were monitored in rapid analysis by an instrumental method, high-performance liquid chromatography (HPLC) coupled with UV detection of the target analytes, AFB1 and ST, upon solvent extraction. Separation of the mycotoxins was achieved on hydrophobic linear alkylsilane stationary phase.
