**3. Discussion**

The development of new express analytical methods that could reduce the time and cost of research is very topical. Novel efficient techniques are urgently needed both for selecting the potential bio-destructors of mycotoxins and for assessing the effectiveness of their action under various conditions. It was shown that cholinesterases and immobilized luminescent cells of photobacteria give a stable analytical signal in the presence of common mycotoxins (Table 1).

The key points for choosing a biological analytical agen<sup>t</sup> for the determination of mycotoxins are LOD and working range. The availability of equipment, the duration of the analysis, the volume of the sample, the need to use additional reagents, and the range of analyzed mycotoxins should also be taken into account. The duration of the analysis (with calibration graph available) was ca. 2–5 min in the case of using enzymes in this work, and 30 min in that of photobacterial cells.

The volume of mycotoxin-containing sample sufficient for the contaminant determination was 10 μL and 4 μL in the cases of cholinesterases or luminescent bacteria, respectively. The analysis of mycotoxins was carried out using generally available laboratory equipment: a spectrophotometer (in the case of enzymes) or a luminometer (in the case of photobacteria). In contrast to the case of luminescent bacteria, the enzyme-based analysis required the use of additional reagents: acetylthiocholine iodide or butyryl-thiocholine iodide was used as substrate and 5,5-dithiobis-(2-nitrobenzoic acid) was used as indicator.

The enzymes were found to be less sensitive to the presence of mycotoxins in the analyzed samples. This was probably due to the fact that in the luminescent cells, in addition to the main analytical luminescent system, there is a large number of interconnected enzymatic systems that are sensitive to the toxicant. Thus, in general, under the conditions of the experiments, the enzymatic analytical systems based on AChE and BChE are somewhat inferior in terms of significant parameters to the cells of photobacteria (Table 1).

Among the studied bacterial strains, despite the high degree of phylogenetic homology, the best analytical characteristics corresponded to *Photobacterium* sp. 9.2 cells (Table 1). The luminescent *Photobacterium* sp. 9.2 and *Photobacterium* sp. 17 cells ensured 44%–83% lower LOD values for zearalenone determination compared to the case of *Photobacterium phosphoreum* B-1717 [17], and for deoxynivalenol and sterigmatocystin the range of working concentrations was 1.5–3.5 times greater in this study. Calibration curves for the quantitative determination of patulin by using photobacterial cells immobilized in poly(vinyl alcohol) cryogel are presented in this work for the first time (Table 1).

The use of the studied biological analytical agents (enzymes and cells) specifically for the direct quantitative determination of mycotoxins in feed is most likely to be inappropriate. The reason is that the solvents that are used to extract mycotoxins into an analytical sample and ensure a high degree of contaminant recovery can themselves be toxic to living cells and enzymes. For an accurate quantitative analysis, in this case, complex sample preparation and standardization for the secondary toxicant are required; in this case, preference is given to liquid chromatography and ELISA [10].

It was shown in this work that the most expedient way to use the photobacteria is the express analysis of mycotoxins, which is an important stage in the assessment of the degree of detoxification of analytical samples due to the destruction of mycotoxins. The analytical samples often contain component toxic to cells other than mycotoxin, e.g., methanol or acetonitrile which are commonly used as extractants-solvents of mycotoxins. In this case it is necessary to account for these additional toxicants when carrying out the calculations, e.g., by appropriate normalization, which was also done in this work.

Despite their somewhat lower sensitivity, enzymes could be used in searching for effective bio-destructors of some mycotoxins. In this case the destruction products are the most predictable, and additional agents can be introduced (if necessary) into the reaction system for directed detoxification of the toxic intermediates of enzymatic mycotoxins' decomposition [5].

The expediency of using the His6-OPH for the destruction of lactone-containing mycotoxins, like zearalenone, was confirmed in the work (Tables 2 and 3). Using immobilized cells of photobacteria as analytical agents, it was shown for the first time that a shift in the pH of the medium from 7.4 to 8.5 when using His6-OPH makes it possible to improve the degree of zearalenone destruction from 93%–94% to 98%–99% (Table 2).

In general, the results obtained are consistent with the literature data on the relatively high hydrolytic activity of lactonases in relation to zearalenone in neutral and slightly alkaline media [17]; the catalytic activity of the enzyme decreases when the pH is lowered [12,18]. The established regularity, allows us to draw certain conclusions about the prospects of creating food supplements based on His6-OPH. The controlled release of this enzyme in the digestive system in the areas with alkaline pH values can ensure efficient decomposition of mycotoxins that enter the body of animals with nutrition under the action of His6-OPH. His6-OPH can be potentially introduced into the feed contaminated with zearalenone, as in the case of recombinant lactonohydrolase [12], for mycotoxin detoxification. Additionally, using His6-OPH in a medium at pH 8.5 provided up to five times faster degradation of zearalenone as compared to recombinant lactonohydrolase expressed in *Penicillium canescence*. This was also demonstrated in this work for the first time.

There is a general consensus that bioanalytical agents such as cholinesterases and photobacterial cells are not selective in their inhibition reactions with various toxins. However, in this work, some obvious preferences were found for bacterial strains, possessing close phylogenetic relations, and cholinesterases in reactions with the same mycotoxins. The best bioindicators in terms of sensitivity and working range (μg /mL) were determined as follows: *Photobacterium* sp. 17 cells for analysis of deoxynivalenol (0.8–89) and patulin (0.2–32); *Photobacterium* sp. 9.2 cells-for analysis of ochratoxin A (0.4–72) and zearalenone (0.2–32); AChE for analysis of sterigmatocystin (0.12–219). Cholinesterases were found to be less sensitive than cells. Calibrations for quantitative determination of patulin using immobilized photobacteria are presented in this work for the first time.

Generally, the use of luminescent cells can significantly reduce the time and financial costs when conducting primary evaluative analyzes of the mycotoxin content in samples during laboratory studies. The efficiency of enzymatic destructors in reactions with mycotoxins can be adequately evaluated using simple equipment and a well-known approach. The information obtained regarding the preferences of bioanalytical agents used for the analysis of a particular mycotoxin will be useful for those researchers who are engaged in the scientific search for bio-destructors of mycotoxins, and simplifies their laboratory screening.

#### **4. Materials and Methods**

#### *4.1. Chemicals and Strains*

Mycotoxins (ochratoxin A, sterigmatocystin, zearalenone, deoxynivalenol, and patulin); cholinesterase enzymes (AChE and BChE); 5,5-dithiobis (2-nitrobenzoic acid), acetylthiocholine iodide, and butyrylthiocholine iodide were purchased from Sigma-Aldrich (St. Louis, MO, USA). For the experiments, concentrated solutions of mycotoxins in methanol were preliminarily prepared. Solutions of mycotoxins of the required concentration were prepared by diluting the original stock solutions of mycotoxins in methanol.

In the analysis, the quenching of the bioluminescence of the immobilized luminous bacteria under the action of the methanol present in the reaction medium was taken into account. Poly(vinyl alcohol) 16/1 (M.w. 84 kDa) was purchased from Sinopec Corp (Beijing, China); peptone and yeas<sup>t</sup> extract were purchased from Difco (Becton, Dickinson and Company, Franklin Lakes, NJ, USA); inorganic salts for Farghaly growth medium and other reagents were purchased from Chimmed (Moscow, Russia). *Photobacterium* sp. 9.2 and *Photobacterium* sp. 17 were provided by A.D. Ismailov (Lomonosov Moscow State University, Moscow, Russia).

#### *4.2. Growth Cells Conditions, Immobilization and Luminescence Measurements*

*Phosphoreum* sp. cells were grown in the Farghaly growth medium and maintained in a submerged culture at 18 ◦C at 60 rpm (IRC-1-U temperature-controlled shaker, Adolf Kuhner AG Apparatebau, Switzerland). The optical density of the culture medium was determined by spectrophotometry at 660 nm (Agilent UV-853 spectrophotometer, Agilent Technologies, Waldbronn, Germany), and the cells were cultivated for 22 h to an optical density of 0.73 ± 0.05, separated from the culture medium by centrifugation (5000 rpm, 15 min, J2 21 centrifuge, Beckman, Brea, CA, USA), and cell biomass used in the immobilization procedure. The procedure for immobilizing the bioluminescent cells in poly (vinyl alcohol) (PVA) cryogel was described previously [20]. The cell biomass was mixed with a 10% ( *w*/*v*) aqueous PVA solution to obtain a 10% ( *w*/*w*) concentration of bacterial cells. This mixture was pipetted into 96-well microplates (0.2 mL/well), which were placed in a freezer at −20 ◦C for 24 h and then thawed at +4 ◦C. The cylinder granules of PVA cryogel (d = 6.6 ± 0.1 mm, h = 4.8 ± 0.1 mm) formed in this way contained cells immobilized by inclusion. The average wet weight of one granule was 0.172 ± 0.001 g.

Luminescence of immobilized bacteria was measured using a 3560 microluminometer (New Horizons Diagnostics Co, Columbia, MD, USA). Luminescence detection was performed in aqueous media based on a 2% NaCl solution at 10 ± 1 ◦C. The maximum level of luminescence (I0) was determined for 10 s at 10 ◦C after thermal equilibration of the flow-through system. For practical purposes, the residual intensity of luminescence was used (I/I0), which was expressed as a percentage of the baseline signal (I0). The residual intensity of luminescence (I/I0) was analyzed in a discrete test after the exposure of the cells to a certain mycotoxin for 0.5 h after its addition to medium containing the analytical agent. The assays were performed in triplicate.

#### *4.3. Mycotoxins Analyses with Cholinesterase Enzymes (AChE and BChE)*

The activity of cholinesterases was determined using the Ellman method [24]. Briefly, 0.96 mL of 0.1 M phosphate buffer (pH 8.0) in a spectrophotometric cell was supplemented with 10 μL of 20 mM 5,5-dithiobis (2-nitrobenzoic acid) in a 0.1 M phosphate buffer (pH 7.0), containing 1.5 g/L Na2CO3. Then, 10 μL of 0.01 mg/mL AChE or 0.2 mg/mL BChE followed by a 10 μL of 0–10 mg/mL mycotoxin in methanol or ethanol was added and vigorously mixed. Reaction was initiated by addition of 10 μL of 50 mM acetylthiocholine iodide or 200 mM butyrylthiocholine iodide for AChE or BChE, respectively.

The rate of formation of 2-nitro-5-thiobenzoic acid at λ = 412 nm was determined using the Agilent 8453 UV-visible spectroscopy system (Agilent Technologies, Waldbronn, Germany). All enzymes, substrates, mycotoxins, and other reagents were freshly prepared before use.

Enzyme activity without any toxins or solvents was monitored, and results were adjusted accordingly. One unit of AChE or BChE activity was defined as the enzyme amount that hydrolyzed 1 μmol of substrate per min at 25 ◦C. The experiments were realized in triplicate.

#### *4.4. Hydrolysis of Zearalenone in Medium with Different pH under the Action of the His6-OPH*

For the experiment, the initial concentration of zearalenone in the reaction medium based on phosphate buffer (pH 7.4 or 8.5) was 65 ± 3 mg/L. The initial toxicity of this

solution was evaluated under the conditions indicated above. The solution of the His6-OPH (0.1 mg/mL) with an activity of 200 U/mL was added to zearalenone solution.

The treatment of the mycotoxin was carried out for 1 h at room temperature without agitation, and the residual toxicity of the obtained solution was verified using immobilized luminescent cells or cholinesterases in a discrete mode of analysis (Table 2).

#### *4.5. Hydrolysis of Zearalenone in Feed Grain Mixture under the Action of the His6-OPH*

Using 0.1 M phosphate buffer (pH 7.5), a solution of zearalenone with concentration 2 g/L was prepared from its concentrated methanol stock solution. The preparation was injected (in the form of a spray) into the feed grain mixture for rats using Classic TiTBiT (Dmitrov, Moscow Region, Russia) at the rate of 10 mg of zearalenone per 1 kg of grain mixture. After that, one half of the grain mixture containing zearalenone was sprayed with a solution of His6-OPH, prepared based on 0.1 M phosphate buffer (pH 7.5), at a dose of 4000 U/kg of the grain mixture, after which the mixture was mechanically stirred and kept for 12 h at 25 ◦C.

The procedure for enzyme production and purification was detailed previously [25]. The activity of His6-OPH was determined as described previously [26], with 7.8 mM aqueous Paraoxon stock solution at 405 nm using the Agilent 8453 UV-visible spectroscopy system (Agilent Technology, Waldbronn, Germany) equipped with a thermostated analytical cell.

After 12 h, the zearalenone concentration was determined in the feed samples with zearalenone, pretreated with the enzyme or without the pretreatment, as well as in the control sample, without any additives. 15 g of each feed grain mixture sample was ground into powder in a laboratory mill and was subject to triple extraction with 40 mL of 84% acetonitrile aqueous solution (*v*/*v*) by mechanical shaker for 15 min. Fractions obtained from each sample were pooled. Then the extract was filtered through paper filters and evaporated to dryness under nitrogen flow. The obtained weighed portion was dissolved in 300 μL of methanol. A total of three samples of methanol stock solution from grain mixture samples were obtained (Table 3). Next, the required dilution of samples was carried out using aqueous buffer solutions to a methanol content of no more than 2%–4%, and the concentration of zearalenone in the samples was determined using cholinesterase enzymes, luminescent cells, or ELISA kit (Table 3).

#### *4.6. Determination of Zearalenone by Enzyme-Linked Immunosorbent Assay (ELISA) Test Kit*

Analyses were carried out using MaxSignal® Zearalenone ELISA Test Kit (Bio Scientific Corp, Austin, TX, USA) with sensitivity 0.3 ng/mL Samples were prepared according to the instructions provided by the manufacturers of the ELISA kits. Optical density was measured at 450 nm using a microplate reader iMark (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
