The potency of bacterial field isolates for zearalenone and aflatoxin B1 detoxification in contaminated crop-origin raw material

Some of saprophytic bacteria are able to inhibit the biosynthesis of mycotoxins, to regulate the spread of microscopic fungi and have prospects of use for preventing contamination of crop-origin raw material by mycotoxins. According to the results of our study we conclude that bacterial field isolates SB10, SB16 and SB20 have the potency to suppress the production of both aflatoxin B1 and zearalenone. Optimal application amount of the culture medium containing 1 × 1010 CFU/mL of bacteria was 5 mL per 1 kg of grain, contaminated with fungi that produce these mycotoxins. However, some of the bacteria-antagonists can also have a negative effect on the processed substrate, and it is necessary to take into account when using the product for the purpose of detoxification.


Introduction
Mycotoxins are toxic secondary metabolites of microscopic fungi. These compounds are very common in nature, are highly resistant to decomposition, their consumption by animals and humans is almost inevitable. Mycotoxins can accumulate in many food and fodder crops in the field, during transportation and storage. The use of contaminated food or feed is the cause of acute and chronic intoxication, mycotoxins have a general toxic, immunosuppressive, mutagenic, teratogenic, carcinogenic, endocrinedestroying effect on living organisms. The global problem lies not only in the negative consequences of direct consumption of plant foods and feeds contaminated with these substances, but also in their ability to pass into animal products, such as meat, milk or eggs. Due to the high resistance of mycotoxins in the environment, the presence of deficiencies in physical and chemical detoxification agents, biotechnological methods using bacteria, yeast, fungi and enzymes are a new strategy for binding or biological decomposition of various mycotoxins [1]. The greatest danger in the toxicological aspect is represented by toxigenic microscopic fungi of the genera Aspergillus and Fusarium [2,3]. Therefore, the main purpose of this study was a comparative assessment of the properties of bacteria aimed at decontamination of mycotoxins produced by fungi Aspergillus flavus and Fusarium sporotrichioides,

Materials and methods
The studies were carried out using toxigenic microscopic fungi Aspergillus flavus and Fusarium sporotrichioides. Previously selected antagonist isolates were used as potential inhibitors of mycotoxin biosynthesis [4,5].
The presence of the ability of isolates to inhibit the biosynthesis of aflatoxin B1 and zearalenone was detected when they were cultured together with the micromycete Aspergillus flavus (aflatoxin B1 producer) and Fusarium sporotrichioides (zearalenone producer) on wheat grain. To do this, the fungi were cultivated for 14 days in a liquid potato medium in a flask under aeration conditions on a mixing device at a rotation speed of 120 rpm and a temperature of 26°C.
Isolates of antagonist bacteria were grown on liquid nutrient media (potato, meat-peptone broth) for 48-72 hours at a temperature of 28-37°C. 100 g of washed wheat grain was placed in 500 ml flasks, 40 ml of tap water was added, thoroughly mixed, kept for 1 hour with frequent shaking and autoclaved for 30 minutes at 1.0 atm. Then 2, 5 and 10 ml of the culture liquid of the antagonist isolate with a titer of 1×10 10 CFU/ml and 1 ml of micromycete suspension at a concentration of 1×10 4 spores/ml were added to the cooled flasks. As a control, flasks with grain treated with 1 ml of micromycete suspension, grain with water and grain with 10 ml of liquid culture medium were used. After thorough mixing, all the flasks were placed in a thermostat and cultured at 26°C.
Mycotoxins were analyzed for 20 days of experience: indication by thin-layer chromatography, identification by high-performance liquid chromatography with tandem mass spectrometric detection. The number of repetitions in each group is 3. During the experiment, the presence of cultures growth was periodically monitored, the rate and nature of micromycete development, the nature of grain changes under the influence of liquid culture medium containing bacterial isolates were evaluated. Statistical processing of the results was carried out using the Microsoft Excel 2010 program. The mycotoxin content was expressed by the mean value (M) and the standard deviation (±SD).

Results
When cultivating the Aspergillus flavus micromycete in flasks with wet grain, full-fledged mycelium development with characteristic grain spoilage was noted. Treatment of the contaminated grain with a culture liquid containing RCA1, PA4, PA7, SB11, SB13, SB22 isolates contributed to the later formation of the substrate mycelium, while the air mycelium was not formed. Against the background of exposure to isolates RPA3, SB15, SB21, SB23, the substrate mycelium of the fungus did not have a pronounced development. Isolates RA8, SB11, SB13 lysed the formed mycelium, and SB10, SB12, SB16, SB20 contributed to the complete absence of pathogen growth. At the same time, the nature of changes depending on the volume of liquid culture medium used was observed, with an increase in the dose, the antifungal activity of the bacterial isolate increased. The use of bacterial isolates RPA3, PA8, SB12, SB22 led to clearly visible damage to the grain, despite the simultaneous inhibition of fungus development. In control flasks with unprocessed grain, the development of micromycetes and changes in the contents were not noted.
Analysis of aflatoxin B1 content in a contaminated micromycete Aspergillus flavus grains showed pronounced efficacy of treatment with liquid culture medium containing bacterial isolates (Table 1). This mycotoxin was not detected in grain treated with SB10, SB12, SB16 and SB20 isolates at concentrations of 5 and 10 ml/kg. Exposure to isolate RCA1 at doses of 2, 5 and 10 ml/kg resulted in a decrease in toxin accumulation in the medium by 73. 5 * <LOD -less than detection limit As can be seen from the presented data, the isolates SB10, SB12, SB16 and SB20 effectively inhibit the biosynthesis of aflatoxin B1, the optimal volume of liquid culture medium application containing 1×10 10 CFU/ml of these bacteria was 5 ml per 1 kg of grain contaminated with a toxigenic strain of the fungus Aspergillus flavus in a concentration of 1×10 4 spores/ml. Nevertheless, the use of SB12 isolate was not advisable due to the deterioration of the substrate treated with this product.
When cultivating the micromycete Fusarium sporotrichioides in flasks with wet grain, full-fledged mycelium development with characteristic grain damage was noted. Treatment of the contaminated grain with a culture liquid containing bacterial isolates RCF1, RPF2, RCF9 and SB17 contributed to the later formation of the substrate mycelium, the air mycelium was not formed. Against the background of exposure to isolates PF7, SB1, SB12, SB15 and SB23, the substrate mycelium of the fungus did not have a pronounced development, RCF8 and RCF11 lysed the mycelium. Isolates SB10, SB12, SB16, SB20 contributed to the complete absence of fungus growth. With an increase in the dose, the antifungal activity of the product containing these bacterial isolates increased. Treatment with PF7, RCF8, SB17 isolates led to grain damage. In control flasks with untreated grain, the development of micromycetes and changes in the contents were not noted.
Analysis of the zearalenone content in grain contaminated with micromycete and treated with culture liquid containing bacterial isolates showed the effectiveness of the use of these preparations ( Table 2). This mycotoxin was not detected in grain treated with SB10, SB12, SB16 and SB20 isolates at concentrations of 5 and 10 ml/kg. Exposure to RPF1 isolate at doses of 2, 5 and 10 ml/kg resulted in a decrease in toxin accumulation in the medium by 67.  * <LOD -less than detection limit As can be seen from the presented data, the isolates SB10, SB12, SB16 and SB20 effectively inhibit the biosynthesis of zearalenone, the optimal volume of application of liquid culture medium containing 1×10 10 CFU/ml of these bacteria was 5 ml per 1 kg of grain contaminated with a toxigenic strain of the fungus Fusarium sporotrichioides at a concentration of 1×10 4 spores/ml. The use of SB12 isolate is not advisable due to damage of the substrate treated with this preparation.

Discussion
To date, physical, chemical and biological methods are used for the biodegradation of mycotoxins [6,7]. Nevertheless, the main conclusion that can be drawn based on many studies on this topic is that most of these approaches are impractical or unsafe, as the nutritional value decreases; production costs increase during the processing and storage of feed, grain; pollution of the environment and its habitat occurs; toxic decomposition products are formed [8,9,10].
Several authors recognize that the most promising method of detoxification of mycotoxins should be based on microbial biodegradation, because bacteria are able to suppress the production of mycotoxins in the mildest way without negative consequences for the environment and its objects [5,11]. The aflatoxin biodegradation by Mycobacterium fluoranthenivorans bacteria, Rhodococcus erythropolis has been reported [12,13,14]. There are studies that suggest that zearalenone can be degraded by Pseudomonas sp., Rhodococcus pyridinivorans and S. cerevisiae [15,16,17,18]. The ability of bacteria of the genus Cupriavidus to biodegradate a number of mycotoxins was revealed [19], and such as aflatoxin B1, ochratoxin A, zearalenone, T-2 toxin and deoxynivalenol were subject to study. According to the research results, seven types of strains can degrade ochratoxin A, four strains can degrade aflatoxin B1, four strains can degrade zearalenone and three strains can degrade T-2 toxin. But none of the strains could destroy deoxynivalenol.
The ability of 42 strains of the genus Rhodococcus to detoxification of aflatoxin B1 and zearalenone was studied [20]. According to the results of studies, 18 of these 42 strains showed the ability to Similarly, in our experiment, there was a difference in detoxification effectiveness when using different field isolates of bacteria. Most likely, these differences are related to the metabolic features of the studied isolates, which were shown earlier [5].
Why do many authors value enzymatic or bacterial detoxification of mycotoxins so much? Because this method combines the advantages of biological and chemical protection, increasing the effectiveness and specificity of this technique.

Conclusion
As a result of the study of the bacteria ability (showing antagonism to the toxigenic microscopic fungi Aspergillus flavus and Fusarium sporotrichioides) to inhibit the biosynthesis of aflatoxin B1 and zearalenone, the effectiveness was shown by bacterial isolates SB10, SB12, SB16, SB20. The optimal volume of application of liquid culture medium containing 1×10 10 CFU/ml of these bacteria was 5 ml per 1 kg of grain contaminated with fungi producing these mycotoxins. The use of SB12 isolate turned out to be inappropriate in view of the damage of the processed grain. Therefore, when screening mycotoxin destructors and micromycete antagonists, along with evaluating the effectiveness, it is necessary to consider the presence of a negative effect on the substrate being treated. Isolates SB10, SB16, SB20 are capable of regulating the spread of microscopic mold fungi, preventing mycotoxicosis and have prospects of application to improve the quality and safety of food raw materials of plant origin.