Exploring the potential of Bacillus subtilis as an additive for decontamination of feed

. Fusariotoxins (deoxynivalenol, nivalenol, T-2 toxin, diacetoxyscirpenol, zearalenone, moniliformin, fumonisins) are secondary metabolites of Fusarium species of fungi that parasitize various plants or are present on them as facultative pathogens. The aim of this work was to evaluate the ability of bacteria that have antimicrobial activity against toxin-producing Fusarium species to detoxify toxic fungal metabolites in order to create additives that improve the quality and safety of feed and other agricultural products. A total of 207 bacterial isolates belonging to various genera and species have been studied. The isolate, designated as RF-45, was characterized by a broad spectrum and a high level of antagonistic activity against most species of Fusarium fungi. Based on morphological-physiological and biochemical studies, analysis of the 16S rRNA gene sequence, the RF-45 isolate was identified as Bacillus subtilis . The bacillary isolate was distinguished by its ability to produce various hydrolytic enzymes (CMCase, avicelasa, β -glucosidase, FPase and xylanase) and antimicrobial metabolites (siderophores, bacteriocins and exopolysaccharides), as well as to detoxify toxic fungal metabolites. It was concluded that B. subtilis RF-45 can be used as an object for creating additives capable of decontaminating feed and other agricultural products.


Introduction
Mycotoxin contamination of agricultural raw materials and products causes colossal damage to the economy of our country [1. 2].The problem of preventing contamination of feed components and other agricultural products with mycotoxins, which pose a serious threat to human and animal health, as well as their decontamination, is of undoubted relevance [3. 4. 5].
The widespread occurrence of Fusarium in nature and the ability to produce a significant amount of mycotoxins, among which, mainly, trichothecene toxins, fumonisins, zearalenones and moniliformins, put them in the category of the most dangerous fungi for agricultural plants, animals and humans [6. 7].The peculiarities of the life strategy of these fungi, the range of various adaptive and protective reactions determine their high aggressiveness, existence in constantly changing and extreme environmental conditions, and the defeat of a wide range of host plants [8. 9].There is evidence that fungi from the genus Fusarium are capable of infecting more than two hundred species of cultivated plants [9. 10].
Among all natural compounds in relation to mammals, fusariotoxins have pronounced hepatotoxic, immunosuppressive, mutagenic and carcinogenic effects [11. 12].The widespread contamination of grain and fodder of plant origin with Fusarium producing mycotoxins leads to various diseases or toxicosis of farm animals, a decrease in productivity and an increase in their mortality [11. 13. 14].It should be noted that the most common species of Fusarium in grain and mixed fodder are F. oxysporum, F. culmorum, F. graminearum, F. solani, F. avanaceum, F. sporotrichioides, F. verticillioides and F. equiseti [5. 11. 13].However, there are currently no effective field chemicals against Fusarium fungi, ways to prevent the production and accumulation of their toxins in the harvested crop, as well as approaches for decontamination of already contaminated products.
One of the main directions of the Strategy of Scientific and Technological Development of the Russian Federation (Decree of the President of the Russian Federation of December 1, 2016 № 642) is the transition to productive and environmentally friendly agriculture, the development and implementation of systems for the rational use of biological protection drugs for agricultural plants and animals.The study of fusariotoxins and the development of approaches to its control in feed products is a rapidly developing area of research on microbial mycotoxins with the practical possibility of using the results obtained in medicine and agriculture [11. 14-20].At present, bacterial antagonists of pathogens of various diseases of agricultural crops form the basis of biological products for reducing the toxicity of forages of plant origin [15. 21. 22].The use of agents that include antagonist bacteria as an active principle is based primarily on the antibiotic mechanism, which regulates the relationship between beneficial and pathogenic microorganisms [23. 24].Nevertheless, the diversity of isolates of phytopathogenic fungi of the genus Fusarium, their activity in certain ecological and geographical zones requires the search for new bacterial strains and the development of effective methods for detoxification of feed and other agricultural products.
The aim of this work was to evaluate the ability of bacteria that have antimicrobial activity against toxin-producing Fusarium species to detoxify toxic fungal metabolites in order to create additives that improve the quality and safety of feed and other agricultural products.

Materials and methods
Materials for the research were 207 bacteria isolated from various natural sources (soil, therapeutic mud, seeds, underground and aboveground parts of agricultural plants, etc.) and obtained from the collection of microbial cultures of the All-Russian Research Institute of Phytopathology (FGBNU VNIIF, Moscow reg., Russia).Toxin-forming molds F. avanaceum, F. culmorum, F. oxysporum, F. moniliforme, F. sporotrichioides, and F. graminearum from the collection of microbial cultures were used as test-isolates.Bacterial and test-isolates were maintained by cultivation in test tubes on slant agar of the following composition (%): glucose -0.63, enzymatic peptone -2.1, sodium chloride -0.65, sodium hydrogen phosphate -0.35, potassium dihydrogen phosphate -0.06, microbiological agar -0.12, at a temperature of (28±1) and (37 ± 1)°C, respectively.
The study of the antagonistic properties of bacterial isolates against test-microorganisms was carried out by the method of delayed antagonism [23].For the formation and diffusion of inhibitory compounds into PDA medium (potato dextrose agar, Merck KGaA, Germany), a two-day culture of the studied strains was sown on its surface with a streak (strip) and cultured at (37±1)°C for 48 h.The test cultures were grown on the same medium at (28±2) °С for 120 h.At the end of the incubation time of bacteria, one disc with the culture of the test fungus was placed at a constant distance between the investigated strain and the disc; cultivation was carried out at (30 ± 1)°C for 3-7 days.As a control of the growth of test cultures, we used parallel seeding on PDA medium without introducing bacterial isolates.The presence and degree of antagonistic activity of bacteria in relation to toxin-producing fungi was assessed by the size of the zone of inhibition of test-isolates (the distance between the border with the growth line of antagonists and the border of the growth of phytopathogens).
Bacterial isolates were identified by studying the cultural-morphological and physiological properties [25].Biochemical identification of the selected bacterial isolate was performed using API 50CH test systems (BioMerieux, France).
Confirmation of the species affiliation of the bacterial isolate was carried out using molecular genetic analysis [26].For amplification of the 16S rRNA gene region, universal primers 27F (5'-GA GTT TGA TCC TGG CTC AG-3') and 1492R (5'-ACG GYT ACC TTG TTA CGA CTT-3') (Evrogen JSC, Moscow, Russia) were used.The PCR product was purified using the PCR Clean Up kit (Thermo Scientific, USA).PCR fragments were sequenced on a genetic sequencer at ZAO «Evrogen» (Moscow, Russia).Comparison of sequenced nucleotide sequences with those known from GenBank (U.S. National Library of Medicine, Bethesda, Maryland, USA) was implemented using the BLASTn system (https://blast.ncbi.nlm.nih.gov/Blast.cgi,National Center for Biotechnology Information, NCBI, USA).
The ability of the selected isolate to produce cellulase-xylanase complex enzymes was determined on a liquid medium of the following composition (g/l): ammonium sulfate -2.5, sodium chloride -0.1, magnesium sulfate heptahydrate -0.125, dipotassium hydrogen phosphate trihydrate -0.25, ferrous sulfate heptahydrate -0.0025, manganese sulfate tetrahydrate -0.025, yeast extract -1.0, carboxymethyl cellulose (CMC) -10.0 [25].The bacterium was incubated on an orbital shaker at 160 rpm under aerobic conditions at a temperature of (32±1)°C for 7 days.Sampling of the culture liquid was carried out every day.The supernatant obtained after separation of bacterial cells from the culture liquid (10000 g, 5 min) was used for further studies on enzyme activity.
Determination of the activity of hydrolytic enzymes in the culture supernatant of the selected bacillus was carried out according to the methods described in [25].
The ability of the selected isolate to produce siderophores and exopolysaccharides was determined on agar media containing chrome azurol S and ruthenium red dyes, respectively [27. 28].
The ability of the selected bacterium to produce bacteriocins against fungi was assessed by the disk diffusion method [29].
Establishment of the ability of bacilli to detoxify toxic metabolites of microscopic fungi was carried out on 30 male Wistar rats, body weight from 180 to 190 g (adaptation time before the start of the experiment was at least 14 days) [15].Animals were divided into 3 equal groups.Individuals of group I (control) received standard feed for 28 days, group IIfeed contaminated with metabolites of toxin-producing fungi F. sporotrichioides and F. oxysporum, group III -toxic feed and additionally culture liquid of the studied bacillus.Animals were kept in the appropriate zoohygienic conditions, had free access to water (without restrictions) and feed (according to daily norms).At the beginning and every 7 days of the study, rats were weighed, their live weight and survival were analyzed.The animals were kept in accordance with the rules adopted by the European Convention for their Protection (86/609 EEC).
The experimental data obtained in the form of tables and diagrams, their statistical processing was carried out using Microsoft Office and Statistica 10.For each sample, the arithmetic mean and their standard errors were calculated.Differences between groups were assessed using the parametric Student's t-test.Differences were considered statistically significant at 95 % confidence level (p ≤ 0.05).

Results and discussion
As a result of the research, 207 bacterial isolates belonging to various genera and species were obtained, characterized by the ability to suppress the growth of toxin-producing species of fungi of the genus Fusarium.According to the degree of inhibition of the growth of testobjects, the isolates obtained by us were divided into 3 main groups: with absent or low (growth inhibition zones up to 5 mm), medium (from 5 to 10 mm) and higher (more than 10 mm) antagonistic action (Figure 1).A comparative analysis of the data obtained showed that a significantly smaller number of bacterial isolates had antagonistic activity against test-fungi.Among bacterial isolates, only 2 of them (0.97 %) suppressed the growth and development of phytopathogenic F. graminearum and 5 (2.4 %) -F.moniliforme.The proportion of bacteria with a relatively high level of fungicidal activity against F. avanaceum, F. culmorum, and F. oxysporum was 6.8, 8.7, and 13.0 %, respectively.
Representatives of Bacillus are classified as promising isolates as the basis of preparations for reducing the toxicity of grain and feed contaminated with mycotoxins.Among them, the bacillary isolate RF-45 had the widest spectrum of antagonistic effect against Fusarium pathogens (Figure 2).Isolate RF-45 was the most active in comparison with other fungi in relation to the causative agent of root rot of plants (the size of the zone of suppression of growth of F. oxysporum was 15.6 ± 0.35 mm).Against the toxin-forming fungi F. avanaceum and F. culmorum, the isolate under study also showed a high level of inhibitory activity (the size of the zone of suppression of fungal growth was 13.9 ± 0.33 and 14.4 ± 0.21 mm, respectively).
Bacterial isolate RF-45 was selected for morphological-physiological, biochemical studies and analysis of the sequence of its 16S rRNA gene (Tables 1-3).The bacterial isolate RF-45 on LA medium formed greyish-white, opaque colonies with irregular margins.The bacterium selected was found in the form of rods with rounded ends, located singly or forming chains, were mobile, formed spores.Bacterial cells after Gram staining looked blue-violet, indicating that the isolate RF-45 belongs to the group of grampositive bacteria.In addition, he was also an aerobe and a moderate thermophile.Determination of the biochemical characteristics of the bacterial isolate RF-45 using the API 50CH test system made it possible to reveal its ability to utilize several organic compounds (substrates) (Table 2).

Carbohydrates
Test results A BLAST search in the GenBank database made it possible to determine that the partial sequence of the 16S rRNA gene of the isolate RF-45 has the highest degree of similarity (98 %) with the corresponding nucleotide sequences of the B. subtilis strain PM48 (accession № OP268482.1)gene, a representative of the Bacillus genus from the Bacillaceae family (Table 3).Thus, according to the analysis of cultural-morphological and physiological-biochemical characteristics and molecular genetic identification, isolate RF-45 was assigned to a representative of the species Bacillus subtilis.

Control
In this work, we studied the dynamics of the activity of hydrolytic enzymes (CMCase, avicelasa, β-glucosidase, FPase and xylanase) during homogeneous submerged cultivation of B. subtilis RF-45 on a medium with carboxymethyl cellulose for 168 h.Analysis of the hydrolase activity of the culture supernatant of the bacillus showed the ability of the latter to produce these enzymes (Table 4).It was established that on the first day of cultivation in the cultural supernatant, B. subtilis RF-45 noted a sharp increase in the activity of hydrolytic enzymes.The most intensive formation and secretion of CMCase, avicelasa and FPase of bacilli into the culture medium occurred at 120 h, while the enzymatic activity was 0.54 ± 0.01, 0.18 ± 0.004 and 0.09 ± 0.002 U/ml, respectively.The maximum activity of β-glucosidase and xylanase was observed at 144 and 96 h of deep cultivation and amounted to 0.02 ± 0.0005 and 4.20 ± 0.09 U/ml, respectively.
The effectiveness of using the culture liquid of B. subtilis RF-45 to reduce the toxicity of food contaminated with microscopic fungi of F. sporotrichioides and F. oxysporum was evaluated.The effectiveness of the culture liquid of bacilli during influence of toxic feed on the body of white rats was assessed by the change in body weight and their survival (Tables 5 and 6).All rats that were fed toxic feed (group II) on the 10th day of the experiment showed clinical signs of toxicosis in the form of decreased appetite, depression, anorexia and diarrhea.By the end of the study, in animals of this group, body weight was lower than control by 11.61 % (Table 5); survival was 80 % (Table 6).The addition of culture liquid B. subtilis RF-45 (group III) to the toxic feed helped to reduce the negative impact of F. sporotrichioides and F. oxysporum metabolites on the live weight gain of rats.In the group of animals that received toxic food treated with bacillus culture liquid, the decrease in their body weight for 28 days was 6.90 % relative to the control group (Table 5); survival was 100 % (Table 6).

Discussion
Fusariotoxins (deoxynivalenol, nivalenol, T-2 toxin, diacetoxyscirpenol, zearalenone, moniliformin, fumonisins) are secondary metabolites of Fusarium species of fungi that parasitize various plants or are present on them as facultative pathogens [9. 30. 31].These compounds produced by Fusarium are among the most dangerous contaminants of agricultural raw materials and food products [5. 11. 12].The use of beneficial bacteria in agriculture can help solve the problem of detoxification of agricultural products [3. 5].It is known that bacteria can degrade such dangerous mycotoxins as fusariotoxins [32][33][34][35][36][37][38].Isolation of new destructor strains is necessary to improve approaches to the biological degradation of toxic fungal metabolites and improve feed safety.In this work, the search for means to reduce the toxicity of feed affected by mycotoxins was aimed at studying bacterial isolates capable of antagonistic activity against Fusarium pathogens -F.avanaceum, F. culmorum, F. oxysporum, F. moniliforme, F. sporotrichioides and F. graminearum.Isolation of microorganisms was carried out from samples of various econiches: medicinal mud, soil with partially degraded deciduous litter, seeds, surface tissues of leaves and roots of various grain crops (wheat, barley and corn), and other sources.Bacteria of the genus Bacillus were represented by the largest number (106 isolates), which is quite natural due to the widespread occurrence of bacilli in nature and their active participation in various processes of ecosystems [39. 40].The resulting bacterial isolates were characterized by active growth and were distinguished by the ability to suppress the growth of test-objects.It should be noted that representatives of the genus Pseudomonas were also classified as promising isolates with biofungicidal properties.In our studies, the spectrum and level of antagonistic activity of isolates of various species and genera did not have a pronounced relationship with the source of their isolation.Nevertheless, most of the highly active bacterial isolates capable of regulating the growth and reproduction of phytopathogens of agricultural crops were isolated from the surface tissues of the aboveground and underground parts of plants.A number of bacterial species that are antagonists of phytopathogenic fungi have been isolated from plant surfaces [41][42][43].Epiphytic bacteria, multiplying on the surface of plants, form a biological barrier that prevents the penetration of pathogens into plant tissues [44].The work of foreign researchers indicate that among epiphytic bacteria, representatives of the genera Bacillus and Pseudomonas have a great antagonistic potential [41].
Among the studied representatives of various genera, B. subtilis isolate RF-45 had the widest spectrum and the highest level of antagonistic activity against most types of toxinproducing fungi of Fusarium (Figure 2).The mechanism of action of B. subtilis strains on phytopathogenic fungi is associated with a complex of factors.These include high growth rate and competitiveness, the ability to form spores, show resistance to extreme conditions for a long time, and also produce biologically active metabolites of various nature: hydrolases (chitosanase, cellulase, glucanase, protease), antibiotic compounds (bacillomycins, mycosubtilins, iturins, surfactins, lipastatins or fengycins), siderophores (bacilbactin), hydrogen cyanide, etc [39. 45-49].Among the biologically active compounds of B. subtilis, cyclic lipopeptides (iturin, surfactin, and fengycin), which are able to act on target cells of phytopathogens at the membrane level, are the main ones when choosing bacterial strains as biological control agents for phytopathogens [39. 45. 46].For the possibility of using selected bacterium in agriculture, it was characterized by biological properties in accordance with the regulated requirements [3. 24].Based on morphological-physiological (Table 1) and biochemical (Table 2) studies, analysis of the 16S rRNA gene sequence (Table 3), the isolate RF-45 was identified as Bacillus subtilis.It was established that B. subtilis RF-45 grew in a wide range of salinity and pH of the environment at temperatures of 15-55 °C (Table 1), which indicated their adaptability to environmental conditions.Under the growth conditions used in our study, this bacterium was able to produce a number of hydrolytic enzymes (CMCase, avicelasa, β-glucosidase, FPase and xylanase) (Table 4), as well as antimicrobial metabolites (siderophores, bacteriocins, and exopolysaccharides) (no results shown).It should be noted that low β-glucosidase activity was detected in the cultural supernatant of the studied bacillus.The process of production of biologically active metabolites by bacilli is primarily determined by the growing conditions (acidity, temperature and aeration), the composition of the culture medium (nutrition sources), the growth phase and the characteristics of their genome, which determines the strain-specific properties [25. 39. 45. 50. 51].The results of our studies are consistent with the data obtained by domestic and foreign authors, who established the ability of B. subtilis to produce extracellular hydrolytic enzymes [25. 39. 46. 52] and compounds with antimicrobial potential [39. 45. 46. 52].Our data open up prospects for the use of the B. subtilis RF-45 in medicine, veterinary medicine, and various industries as a producer of enzymes and immunoactive factors, as well as the active basis of biopreservatives, probiotics, synbiotics, metabiotics, and feed additives.
In our studies, in addition to morphological-physiological and biochemical studies of B. subtilis RF-45, under laboratory conditions, we assessed its ability to reduce the toxicity of feed contaminated microscopic fungi F. sporotrichioides and F. oxysporum.In earlier studies, we have shown the ability of certain strains of microorganisms, including B. subtilis, to potential adsorption and enzymatic detoxification of fusariotoxins in mixed feed [15].Bacillus strains, including B. subtilis, transform toxins produced by various species of the Fusarium genus into less dangerous or non-toxic metabolites for humans and animals [32. 33. 53-58].Despite the fact that many bacillary strains have toxin-degrading activity, their use for detoxification of feed and other agricultural products is limited.The limitations, most likely, may be associated with insufficient knowledge of the problem of the safety of the use of bacilli in agriculture [3].An analysis of literature data indicates the scale of the use of B. subtilis for obtaining various products from bacterial biomass or their biologically active metabolites [39. 40. 52. 59].The Food and Drug Administration (FDA), agency of the U.S., has awarded B. subtilis strains the status of GRAS (Generally Regarded as Safe), which is a prerequisite for their widespread use [59].However, B. subtilis may contain genes responsible for the synthesis of pathogenicity factors, namely enterotoxins [3].In the present study, we have shown that the treatment of feed contaminated with metabolites of the microscopic fungi F. sporotrichioides and F. oxysporum with the culture liquid of B. subtilis RF-45 contributes to a decrease in its toxicity (Table 5), as well as a protective effect on the animal organism (Table 6).Thus, B. subtilis RF-45 has the ability to detoxify toxic metabolites of microscopic fungi and can potentially be used to improve feed quality and safety.However, the use of a destructor bacterium has certain limitations, which are due to its possible negative impact on the quality of processed products.In this regard, future studies will be aimed at searching for metabolites of the strain with mycotoxin-degrading activity and creating preparations based on them that can combat contamination of feed and other crop products.

Conclusions
As a result of the research, the most active strain of the Bacillus subtilis species was isolated and identified.It is characterized by a wide spectrum of antagonistic activity against toxinproducing Fusarium species, produces various biologically active compounds and is capable of detoxifying toxic metabolites of microscopic fungi, which allows us to recommend it for use as the basis of a drug to improve the quality and safety of feed and other agricultural products.

Table 1 .
Morphological, cultural and physiological properties of bacterial isolate RF-45.

Table 3 .
Identification of the selected bacterial isolate RF-45.

Table 4 .
Maximum activity of hydrolases of B. subtilis RF-45 on the medium with carboxymethyl cellulose.

Table 5 .
Dynamics of the body weight of Wistar rats with mycotoxicosis against the background of the use of the culture liquid of B. subtilis RF-45

Table 6 .
Survival of Wistar rats with mycotoxicosis against the background of the use of culture liquid of B. subtilis RF-45