**2. Results**

#### *2.1. The Quantitative Express-Analysis of Mycotoxins in Liquid Media Involving Cholinesterases or Immobilized Bioluminescent Photobacterial Cells*

It was shown with sufficiently good reproducibility, that cholinesterases and immobilized luminescent photobacterial cells can be successfully used to perform the quantitative express-analysis of at least one of five mycotoxins (deoxynivalenol, ochratoxin A, patulin, sterigmatocystin, and zearalenone) in liquid media in discrete mode (Table 1 and Figures 1–3).

**Table 1.** Analytical characteristics of different mycotoxins' assay (Figures 1–3) based on application of cholinesterases (Figures 1 and 2) or immobilized luminescent cells (Figure 3) and linearization equations (R<sup>2</sup> is adjusted coefficient of determination).


Activity = a − bx lg (concentration, μg/mL); 2 Activity = a − bx (concentration, μg/mL).

1

> Calibration plots for quantitative analysis are presented in Figures 1–3 in the coordinates, in which the obtained data can be successfully linearized. Enzymes were found to be less sensitive than photobacteria to the presence of mycotoxins. In general, a shift in the range of working concentrations upward was noted for enzymes. Both enzymes showed lower sensitivity to the presence of patulin as compared to cells.

> AChE was more sensitive to ochratoxin A and sterigmatocystin and less sensitive to DON as compared to BChE. According to the results obtained, the adjusted coefficient

of determination (R2) for both enzymes was close to 1 (Table 1), whereas for luminescent bacterial cells, mainly for the *Photobacterium* sp. 17, the values of R<sup>2</sup> below 0.97 were obtained. Note that calculated R<sup>2</sup> values exceeding 0.9 indicate the possibility of using the corresponding tool (both enzymes and photobacteria in our case) for analytical purposes.

The results obtained allow the selection of the most acceptable analytical method for an express analysis of each mycotoxin in terms of the working concentration range and the lower limit of detection (LOD) value.

Thus, *Photobacterium* sp. 17 cells provide the lowest LOD and a fairly wide working concentration range for the analysis of deoxynivalenol, whereas *Photobacterium* sp. 9.2 ensure the best set of parameters for ochratoxin A and zearalenone detection, *Photobacterium* sp. 17 cells are optimal for patulin, and AChE is the instrument of choice for sterigmatocystin.

**Figure 1.** Inhibition effect of deoxynivalenol, patulin, ochratoxin A, sterigmatocystin, and zearalenone on acetylcholinesterase (AChE) activity. Activity was measured by Ellman assay with acetylthiocholine iodide as substrate in 0.1 M phosphate buffer (pH 8.0).

**Figure 2.** Inhibition effect of deoxynivalenol, patulin, ochratoxin A, zearalenone, and sterigmatocystinon on butyrylcholinesterase (BChE) activity. Activity was measured by Ellman assay with butyrylthiocholine iodide as substrate in 0.1 M phosphate buffer (pH 8.0).

**Figure 3.** Residual intensity of luminescence of immobilized *Photobacterium* sp. 9.2 (**a**) and *Photobacterium* sp. 17 (**b**) cells in the presence of various mycotoxins (DON—deoxynivalenol, ZEA—zearalenone, SCN—sterigmatocystin, OCH—ochratoxin A, PAT—patulin) in discrete analysis.

#### *2.2. Assessment of Toxicity of the Reaction Medium Obtained after Hydrolysis of Zearalenone by His6-OPH in the Media with Different pH*

Using acetyl-cholinesterases, photobacterial cells, and ELISA Test Kit as analytical tools, we have proven the possibility of zearalenone destruction under the action of His6- OPH in a liquid medium at different pH values at an initial mycotoxin concentration of 65 ± 3 μg/mL (Table 2). The results of this study agree with those we obtained earlier [17]. It was noted that the low sensitivity of enzymes allowed the detection of only the initial concentration of mycotoxin in the test solution. The residual concentration of zearalenone after the action of His6-OPH in the case of enzymatic analytical agents could only be determined using BChE for a sample with pH 7.4 (Table 2). Photobacterial cells, however, ensured accuracy high enough to reliably assess the degree of zearalenone destruction under the action of His6-OPH for different medium pH values. The accuracy of mycotoxin detection with photobacteria was in fact found to be at least as high as that ensured with the ELISA Test Kit.

**Table 2.** The residual zearalenone concentrations in the media with hexa-histidine-tagged organophosphorus hydrolase (His6-OPH) after 1 h of enzymatic treatment. The initial zearalenone concentration was 65 ± 3 μg/mL.


\* DH is the degree of zearalenone hydrolysis = the percentage of the hydrolyzed zearalenone concentration in relation to its initial level; -: samples were not analyzed.

#### *2.3. Zearalenone Biodegradation in Feed Grain Mixture under the Action of the Enzyme His6-OPH*

In a model experiment using enzymes, photobacterial cells, and ELISA Test Kit as analytical agents, it was possible for the first time to assess detoxification of food raw materials (feed grain mixture) initially contaminated with zearalenone at a concentration of 10 mg/kg. Feed was treated with His6-OPH enzyme to push the toxin concentration below the levels specified in the generally accepted quality standards, so that the resulting contaminant concentration was below 1 mg/kg (Table 3).

**Table 3.** Residual concentrations of zearalenone in the feed grain mixture (initial contamination of feed grain mixture by zearalenone was 10 mg/kg) after its treatment with enzyme His6-OPH (ED) during 12 h and without it (NE).


\* R is the degree of zearalenone recovery; the percentage of the NE zearalenone concentration to its initial concentration; \*\* D is the level of zearalenone detoxification in feed; the percentage of the change in zearalenone concentration in feed grain mixture as the result of enzymatic destruction (NE-ED) to the non-enzymatic degradation (NE); \*\*\* There was no inhibition of BChE activity.

> In the case of enzymatic analytical agents, only BChE allowed the determination of the concentration of zearalenone in the analytical sample of the raw material with the maximum concentration of zearalenone (Table 3). The use of biological analytical agents resulted in slightly higher values of toxicant concentration than in case of using the ELISA Test Kit. This was probably due to the presence of substances other than zearalenone, which reduce the activity of enzymes in the analytical samples. These substances could

have originated from the feedstock and been transferred together with zearalenone during the extraction stage. It is important that the calculated detoxification degree was similar in the case of photobacterial cells and ELISA Test Kit (Table 3).

Thus, photobacteria can be efficiently used as analytical agents for evaluating the detoxification of real raw materials. They can greatly facilitate the search for bio-destructors of any target mycotoxin, as well as the assessment of the efficiency of detoxification using various biological agents. It was shown that an acetonitrile-based extractant can be recommended for the extraction of zearalenone from the feedstock during sample preparation. This extractant provides a high degree of recovery of this toxicant (Table 3), which is consistent with the previously published data [22,23].
