Antifungal Activity and Mycotoxin Degradation Potential of Bioprotective Microorganisms to Application in Animal Food Production Chain

1. Introduction

Animal food production has increased dramatically in the last decade. Considering only meat industry, in 2012 the world produced 245.73 million tons; in contrast, 2021 data resulted in a production of 357.39 million tons, which means an increase of 111.66 million tons in just nine years. Among all meat commodities, pork and poultry stand out, accounting for 33.92% and 38.89% of world production, respectively [1].

One of the major problems affecting the animal food production chain is contamination by mycotoxins. These mycotoxins can cause production losses, since there is a decrease in the zootechnical indexes of the animais, and also cause harm to consumers due to their residues in animal-source foods, once their chemical characteristics make them resistant to the main preservation methods, such as freezing and cooking [2]. Mycotoxins can reach the animal food production chain through the use of contaminated raw materials in the preparation of animal feed, or through fungal contamination in feed stored in an inadequate situation. Research indicates that between 30 and 100% of foods and feed intended for human and animal consumption have some degree of fungal and/or mycotoxin contamination [3].

Among the most studied mycotoxins, fumonisin B1 (FB1) and zearalenone (ZEA) present important impact on both human and animal health. FB1 is a human possibly carcinogenic mycotoxin – group 2B by the International Agency for Research on Cancer (IARC) –, related to esophageal cancer and neural tube defects during embryogenesis. ZEA is a toxin with strong estrogenic activity – group 3 by the IARC –, related to precocious puberty and development disorders, especially in women. Additionally, there are evidence of immunotoxic, hepatotoxic, nephrotoxic, genotoxic, and hematotoxic effects [4].

The search for strategies to control fungal contamination and the consequent production of mycotoxins is constant, and bacterial biocontrol has been highlighted in recent years, with several research being developed in the area [5,6,7,8]. Considering that fungal and mycotoxin control is essential throughout the entire food production chain, and that animal-foods can be vehicles of these contaminants for humans, the objective of this research was to evaluate the antifungal and antimycotoxigenic potential of different microorganisms, focusing on food-animals production.

2. Materials and Methods

2.1. Strains and Materials

Twenty-three potentially bioprotective microorganisms and thirteen filamentous fungi were used, obtained from different microbiological collections (Table 1). The bacteria and yeasts were maintained in culture media + 20% glycerol, and the fungi in culture media + 30% glycerol, both at -80 ºC until use, and were reactivated in the same culture media, incubated at 37 ⁰C for 24 - 48h (bacteria and yeast) or at 25 ⁰C for 5 - 7d (fungi), with at least two passages prior to the analyses.

The bacteria and yeasts were chosen for the bioprotective potential presented by microorganisms of the same species, according to research available in scientific databases, and the fungi for their importance in food and feed contamination, and for their presumptive mycotoxin production. The taxonomy of Lactobacillaceae was actualized according to Zheng et al [9].

All culture media used were purchased from Oxoid (Hampshire, United Kingdom). All chemicals and reagents used were analytical or chromatographic grade, purchased from Fisher Scientific (Hudson, NH) and Sigma-Aldrich (St. Louis, MO). Ultra-pure water (~18.2 MΩ/cm resistivity) was obtained from a Milli-Q purification system (Merck Millipore, Darmstadt, Germany).

2.2. Degradation of Mycotoxins in Culture Media

Initially, a mycotoxin degradation potential screening was conducted for the putatively bioprotective microorganisms. Bacteria and yeasts were acclimated for growth in a minimal medium (consisting of 5.0 g/L of tryptone, 1.0 g/L of glucose, and 2.5 g/L of yeast extract) for a minimum of two passages. Following adaptation, the medium was augmented with 1 µg/mL of ZEA or FB1, and was then inoculated with 1% of a fresh culture of each microorganism under examination, individually analyzed (adjusted to a 0.5 McFarland Standard, approximately ~1.5 × 10⁸ CFU/mL). The tubes were incubated at 37 ⁰C for 48h with agitation at 120 rpm. Subsequent to incubation, the content was filtered using a nylon syringe filter (0.22 µm pore size) and subjected to ultra-high performance liquid chromatography coupled with time-of-flight mass spectrometry (UHPLC-MS/qTOF) for quantification of ZEA and FB1. Control tubes included minimal medium alone (negative control) and minimal medium supplemented with 1 µg/mL of ZEA or FB1 (positive control) [10].

Chromatographic analysis was carried out using an Agilent 1200 HPLC system (Agilent Technologies, Santa Clara, CA), equipped with an auto-sampler, vacuum degasser, and binary pump. Analyte separation was achieved with a Gemini C18 column (50 mm × 2 mm, 110 Å, 3 μm particle size) sourced from Phenomenex (Palo Alto, CA). The mobile phases consisted of water with 0.1% formic acid (solvent A) and acetonitrile with 0.1% formic acid (solvent B), with a flow rate of 0.3 mL/min in a gradient (0 min: 5% B; 30 min: 95% B; 35 min: 5% B), and a total analysis run time of 35 minutes. The injection volume was 5 µL [11].

For mass spectrometry analyses, an Agilent Ultra High-Definition Accurate Mass MS/qTOF system (6540, Agilent Technologies, Santa Clara, CA) was utilized, coupled with an Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI, Agilent Technologies, Santa Clara, CA) interface operating in positive ion mode. The optimized mass spectrometry parameters included a fragment voltage of 175 V, a capillary voltage of 3.5 kV, collision energies of 10, 20, and 40 eV, a nebulizer pressure of 30 psi, a drying gas flow of 8 L/min (using N2), and a temperature set at 350 ⁰C. Data analysis was conducted using MassHunter Qualitative Analysis Software B.08.00 (Agilent Technologies, Santa Clara, CA) [11].

Following the initial screening of degradation activity, the microorganisms exhibiting a degradation rate of ≥90% were subjected to further assessments under the following conditions (analyses for FB1 were not conducted as the minimum degradation threshold was not met in the initial screening): i) the microorganisms were inoculated at a concentration of 10⁴ CFU/mL in the presence of 1 µg/mL of ZEA, and incubated for 48h at 37 ⁰C; ii) cell-free supernatants of each strain were obtained after a 48h-bacterial growth, followed by centrifugation (3500 × g, 10 min) and filtration using a nylon syringe filter (0.22 µm pore size). These supernatants were then incubated with 1 µg/mL of ZEA for 48h at 37 ⁰C; iii) intracellular metabolites of each strain were obtained after rupturing the bacterial biomass's plasmatic membrane in a hypotonic solution (ultrapure water) and subjecting it to freezing cycles (-45 ⁰C and 37 ⁰C, 2h/cycle, repeated 4 times). The resulting solution was then subjected to centrifugation (3500 × g, 10 min), and the supernatant was collected. Subsequently, it was incubated with 1 µg/mL of ZEA for 48h at 37 ⁰C; iv) autoclaved culture after a 48h-bacterial growth was incubated with 1 µg/mL of ZEA for 48h at 37 ⁰C; and v) to evaluate the adsorptive potential, the recovery of ZEA from bacterial biomass after incubation for 48h at 37 ⁰C in the presence of 1 µg/mL of ZEA was conducted. The degradation of ZEA, the adsorption potential of the strains, and the formation of degradation compounds, were evaluated using UHPLC-MS/qTOF, as described previously.

2.3. Inhibition of Fungi and Mycotoxins Production in Culture Media

The bioprotective microorganisms were cultivated in a liquid culture medium for 24h at 37 ⁰C. Subsequently, 3 µL of the fresh culture were inoculated at the center of potato dextrose agar (PDA) plates. After drying, fungal explants with a diameter of 0.5 cm were placed at the edges of the plates. These explants were derived from recent fungal cultures that were initially prepared on PDA plates and incubated at 25 ⁰C for 5 - 7d, until sporulation occurred. Plates containing both bioprotective microorganisms and fungal explants were incubated at 25 ⁰C for 7d, and the extent of fungal growth was measured at 7d. Control plates contained only fungal explants (positive control) or only bioprotective microorganisms (negative control) [12].

Following the measurement, the agar was entirely removed from the plates and soaked in 15 mL of methanol to extract the mycotoxins. The flasks were agitated at 150 rpm for 24h, and then the methanol was filtered through a nylon filter with a porosity of 0.22 µm. Subsequently, the filtered methanol was subjected to UHPLC-MS/qTOF analysis, as described previously, for the quantification of mycotoxins and the detection of degradation compounds [13].

2.4. Characterization of Antifungal and Antimycotoxigenic Metabolites of Bioprotective Microorganisms

The selected bacteria (≥90% ZEA degradation in initial screening) were inoculated into 200 mL of tryptone soy broth (TSB) at a concentration of ~10⁴ CFU/mL. The culture was maintained at 37 ⁰C on an orbital shaker operating at 120 rpm. At specific time points (0, 24, 48, and 72h), aliquots were extracted, subjected to centrifugation at 3500 × g, diluted with Milli-Q water at a ratio of 1:5 (v/v), and then filtered using a nylon syringe filter with a 0.22 µm pore size. To prevent any potential interference with the growth process, separate samples were prepared for each of the specified time points [14]. These samples were subsequently analyzed using UHPLC-MS/qTOF as described previously.

Upon the identification of metabolites, additional samples were collected for lyophilization, performed using a Lab Freeze Dryer (OLT-FD-10N, Xiamen Ollital Technology Co., Ltd.) to antimicrobial activity analysis.

2.5. Antimicrobial Activity of the Lyophilized Metabolites

The antimicrobial activity of metabolites was conducted in two ways, both in solid and liquid media, involving the inhibition of fungal growth and mycotoxin production on solid media, and the determination of minimum inhibitory and fungicidal concentrations in liquid media.

For the analysis of growth inhibition zones, 100 µL of a spore suspension containing 10⁴ spores/mL were inoculated onto the surface of PDA plates. After complete drying, spots were made on the plate, and were added the lyophilized metabolites reconstituted in ultrapure water at a concentration of 500 g/L. The plates were incubated at 25 ⁰C for 3 - 7d, and the inhibition of fungal growth was measured. The negative control contained only ultrapure water, while the positive control consisted of PDA plates with fungal inoculum [15].

To determine the minimum inhibitory concentration, concentrations ranging from 0.98 to 500 g/L of lyophilized metabolites reconstituted in TSB were used, employing the microdilution technique. The wells contained 5 × 10⁴ spores/mL with a final volume of 200 µL. The positive control contained only the spore suspension, and the negative control contained sterile TSB. Plates were incubated at 25 ⁰C for 48h, and the lowest concentration exhibiting visible inhibition of fungal growth was determined as the minimum inhibitory concentration. After reading, the contents of wells with no visible growth were inoculated onto PDA plates. The lowest concentration required to kill the fungus was defined as the minimum fungicidal concentration [16].

2.6. Zearalenone Degradation in Swine and Poultry In Vitro Digestion

For the simulated digestion analyses, encapsulated bacteria from the same batch produced for previous research conducted by our research group were used. Details regarding the encapsulation parameters, efficiency, and bacterial population of the resulting product can be found in the Supplementary Material. Before the digestions, the capsules were resuspended in sterile ultrapure water at a concentration of 50 g/L until complete homogenization and rehydration.

Swine digestion simulation was conducted in accordance with Evangelista et al [17] with modifications. One milliliter of the capsule suspension, contaminated with 1 µg/mL of ZEA, were utilized. In tubes containing the samples, 20 mL of phosphate buffer pH 6.0, 8 mL of 0.2 M HCl, and 50 U of pepsin were added. The pH was adjusted to 2.0±0.2, and the material was incubated for 2h at 39 ⁰C with agitation at 250 rpm (stomach phase). After incubation, 7 mL of phosphate buffer pH 6.8, 4 mL of 0.6 M NaOH, and 100 mg of pancreatin (4X USP) were added. The pH was adjusted to 6.8±0.2, and the material was incubated for 4h at 39 ⁰C with agitation (intestinal phase). Samples were collected before and after the stomach phase, and after the intestinal phase, for the quantification of mycotoxins and degradation metabolites using UHPLC-MS/qTOF, as described previously.

Poultry digestion simulation was conducted as described by Evangelista et al [17] with modifications. One milliliter of the capsule suspension, contaminated with 1 µg/mL of ZEA, were used. In tubes containing the samples, 10 mL of sterile deionized water was added, and the pH was adjusted to 6.0±0.2. Incubation with agitation at 250 rpm at 41 ⁰C for 30 minutes was performed (crop phase). After this period, 25 mL of sterile deionized water and 134,750 U of pepsin were added, with the pH adjusted to 2.5±0.2. The material was incubated at the same temperature for another 30 minutes with agitation (proventriculus phase). Glass beads with a diameter of 0.5 cm were added to simulate mechanical digestion in the gizzard, and the pH was adjusted to 3.5±0.2, with another incubation for 1h with agitation (gizzard phase). Then, 280 U of pancreatin and 135 mg of bile salts were added, and the pH was adjusted to 6.2±0.2, with another incubation for 2h with agitation (small intestine phase). Finally, the pH was adjusted to 7.0±0.2, and the material was incubated for 20 minutes with agitation (large intestine phase). Samples were collected before the crop phase, after the proventriculus phase, and after the large intestine phase, for the quantification of mycotoxins and degradation metabolites by UHPLC-MS/qTOF, as previously described.

2.7. Statistical Analyses

The results are presented as mean ± standard deviation. Statistical analysis was performed using GraphPad Prism 8.0 (San Diego, CA). Data were assessed for normality using the Shapiro-Wilk test, and an analysis of variance by ANOVA was conducted, followed by the Tukey test. The significance level was set at p<0.05.

3. Results

3.1. Screening of Degradation Potential and Effect of the Selected Strains

In the screening for ZEA degradation potential, only three strains showed no activity, and degradation rates ranged from 0.00 to 96.75%. In contrast, in the analysis with FB1, all strains exhibited some level of activity, ranging from 0.33 to 37.13%. Significant variability in degradation potential was observed, even among strains of the same species, as seen between Bacillus amyloliquefaciens CECT 493 and Bacillus amyloliquefaciens plantarum MLB3, which exhibited degradation of ZEA of 0.00% and 93.09%, respectively. For ZEA degradation, four strains demonstrated effectiveness within the established methodological criteria and were selected for further testing: B. amyloliquefaciens plantarum MLB3, Bacillus subtilis MLB2, Bacillus velezensis CL197, and Streptomyces griseus CECT 3276. Satisfactory FB1 degradation was not achieved with the applied methodology; therefore, further tests with this mycotoxin were not pursued (Table 2). Screening was also conducted for ochratoxin A degradation; however, none of the bacteria exhibited any degree of degradation (unpublished data).

The selected bacteria exhibited varying degradation rates when evaluated by the five established methodological parameters. The most effective degradation was achieved when using intracellular metabolites, resulting in a reduction of 89.34±0.72% to 92.93±2.17%. When assessing ZEA adsorption, low levels were observed, ranging from 10.10±0.68% to 18.59±4.80%. The combination of low adsorption with high degradation is a highly desirable characteristic since adsorption can be reversed along the gastrointestinal tract in potential in vivo applications, thereby restoring ZEA toxicity, whereas degradation is associated with a permanent modification in the toxin's structure (Table 3).

When assessing the metabolites produced after the inoculation of each bacterium with ZEA, the predominant presence was ZEA with conjugates, such as glucosides, acetyl, malonyl, or sulfate. Additionally, zeranol and zearalenol were observed in their original forms or as conjugates. The formation of conjugates can pose a concern, as they may potentially separate within the organism, allowing the toxin to exert its deleterious effects. Furthermore, zeranol and zearalenol are compounds that may exhibit increased toxic activity based on their chemical structure. However, it should be noted that the concentration of metabolites remained relatively low. While the analysis began with 1 µg/mL of ZEA, the concentration of metabolites ranged from 11.20±0.87 to 99.10±0.93 ng/mL (Table 4).

3.2. Antifungal and Antimycotoxigenic Activity of the Selected Bacteria in Solid Culture Media

When fungi and bacteria were co-inoculated in solid media, all bacteria were able to inhibit the growth of at least one fungal species. The best result was observed with B. amyloliquefaciens plantarum MLB3, which exhibited inhibitory activity against 100% of the fungi (13 strains), while the lowest result was observed with S. griseus CECT 3276, showing only 7.69% effectiveness (1 strain). This difference may be attributed to the considerably slower growth of S. griseus CECT 3276 in solid media compared to other bacteria, allowing the fungi more time to develop and occupy the available growth space. The strains B. velezensis CL197 and B. subtilis MLB2 exhibited 84.62% (11 strains) and 15.38% (2 strains) effectiveness, respectively (Table 5). Although the fungi were selected for their documented ability to produce mycotoxins, no production was detected in either the control or treatment groups. This may be due to routine cultivation in a nutrient-rich medium where there is no stimulus for the production of secondary metabolites responsible for the microorganism's defense and survival.

3.3. Bacterial Metabolites Characterization and Antifungal Effects

Among the evaluated bacteria, it was possible to provide a better characterization of the antifungal metabolites generated by B. velezensis CL197, with the detection of Fengycin C, Gageostatin B, Gageostatin C, Marihysin A, Plipastatin A2, and Plipastatin B1. For the bacterium B. amyloliquefaciens plantarum MLB3, the metabolites Gageostatin C and Marihysin A were identified and quantified (Table 6). No metabolites were detected in the cultures of B. subtilis MLB2 under the conditions used in this study. A total of 8 metabolites with potential antifungal activity were detected in the cultures of S. griseus CECT 3276. However, for their identification, a database common to metabolites produced by bacteria of the Streptomyces genus was employed. Given that this genus is widely recognized for its production of metabolites, either through natural pathways or by induction/bioengineering, the results presented here may be compromised. The compounds identified could potentially correspond to similar ones not specifically indicated, such as isomers (Table 7).

In the liquid culture medium inhibition test, the metabolites produced by B. amyloliquefaciens plantarum MLB3, B. subtilis MLB2, and S. griseus CECT 3276 for 24h did not exhibit fungistatic or fungicidal effects. However, the metabolites produced by B. velezensis CL197 demonstrated fungistatic effects at concentrations ranging from 250 to 500 g/L. On the other hand, when utilizing metabolites produced for 48 and 72h, all of them were effective, leading to fungal inhibition at concentrations of 250 and 500 g/L, and fungicidal activity at a concentration of 500 g/L (Table 8).

When evaluated in solid culture medium, only the metabolites generated through 72h of incubation exhibited inhibitory activity at a dose of 500 g/L. Notably, the metabolites produced by B. velezensis CL197 and S. griseus CECT 3276 demonstrated inhibitions ranging from 5.5±0.1 to 6.2±0.2 mm and 5.2±0.2 to 6.2±0.6 mm, respectively. The inhibition generated by the metabolites of B. amyloliquefaciens plantarum MLB3 was slightly lower, ranging from 3.2±0.9 to 4.0±0.2 mm. On the other hand, the metabolites of B. subtilis MLB2 were effective in only 6 out of the 13 fungal strains evaluated, with inhibition ranging from 1.1±0.1 to 1.4±0.3 mm (Table 9).

3.4. Mycotoxin Degradation in Simulated Digestion

All bacteria exhibited effective ZEA degradation throughout the simulated digestive process. In the swine digestion simulation, B. subtilis MLB2 demonstrated remarkable efficacy, achieving complete toxin degradation after a 2h-incubation. In the poultry simulation, notable performances were observed for B. velezensis CL197, B. subtilis MLB2, and B. amyloliquefaciens plantarum MLB3, with each achieving 100% degradation, or statistically equivalent levels, within a 2h-digestion period (Table 10).

When evaluating the production of metabolites in simulated digestion, the same profile as observed in the culture media degradation was obtained. The metabolites mainly consisted of conjugates, all with concentrations at least 10 times lower than the initial ZEA concentration. In poultry digestion, the concentrations of metabolites ranged from 2.17±0.84 to 68.74±0.44 (Table 11), and in swine digestion, they ranged from 3.21±0.29 to 258.32±0.36 (Table 12). During in vitro simulations, the reversion of conjugates to their original condition was not observed. However, in vivo tests are required for a more in-depth evaluation, as there are various factors in the animal organism that may facilitate the reconversion of conjugates into ZEA.

4. Discussion

Initially, during the screening for ZEA and FB1 degradation, significant variability in results was observed, even among strains of the same species. This variation is associated with the intrinsic characteristics of each strain, such as metabolite production, enzyme activity, etc. In the present study, none of the strains were capable of degrading FB1 satisfactorily. However, due to their mild performance, they may be subjects of future research, involving an in-depth assessment of their mechanisms of action. This would help determine the feasibility of enhancing their effectiveness through molecular and bioengineering techniques. During the screening for ZEA degradation, four bacteria stood out: B. amyloliquefaciens plantarum MLB3, B. subtilis MLB2, B. velezensis CL197, and S. griseus CECT 3276. Previous research had already demonstrated the potential of these species in ZEA control. Lee et al [37] showed that B. amyloliquefaciens completely degraded 3.5 μg/mL of ZEA in 24h of incubation. Lei et al [38] utilized a B. subtilis strain for ZEA degradation and achieved positive results, with approximately an 89%-reduction in the initial toxin concentration. Wang et al [39] used a B. velezensis strain and achieved 100% ZEA degradation with an initial concentration of 7.45 μg/mL after 72h of incubation. While recent research using S. griseus was not found, Harkai et al [40] demonstrated that Streptomyces species had the potential for ZEA degradation, with efficiency ranging from 87.85±6.68 to 99.64±0.23% at an initial concentration of 1 μg/mL of ZEA.

During the evaluation of different potential degradation mechanisms and the possibility of adsorption, it was observed that the most efficient form of utilization was the intracellular metabolites, followed by the use of the whole bacterial culture. A low adsorption rate, as mentioned earlier, is a desirable characteristic since adsorption can be reversed throughout the gastrointestinal tract. In this analysis, the most abundant ZEA metabolites were conjugates, albeit with a drastic reduction in their concentration compared to the initial ZEA concentration. This demonstrates that the mycotoxin was predominantly biotransformed, necessitating further in-depth tests to specifically determine the exact metabolization products. Notably, it is observed that compounds of higher toxicity, such as α-zearalenol, were either not formed or present in extremely low quantities relative to the initial ZEA concentration. This indicates that, even without a complete characterization of the degradation products, no compounds that could increase the risks associated with ZEA consumption were formed. The tests revealed that efficient degradation requires either an active bacterium or its metabolites, which exhibit a thermolabile nature, since when autoclaved culture was used (primarily with broken cells), the degradation rate decreased significantly. Considering the industrial process's stages, the most practical application involves using the complete culture without the need for further manipulation. Hence, this method was chosen for subsequent tests, as it still achieved acceptable degradation levels, facilitating the future development of biotechnological products for application in the productive sector.

The evaluated bacteria, besides showing potential for ZEA degradation in culture medium, also demonstrated the ability to inhibit fungal growth. This characteristic can be particularly interesting since, in addition to mitigating the effects of already-produced toxins, they can prevent fungal development, which leads to the subsequent production of mycotoxins. Their bioprotective activities were assessed against different types of fungi, producers of various mycotoxins, and at least one strain proved effective against each of the fungi. This demonstrates their wide applicability in this role, with the possibility of developing combinations between them to enhance results. No mycotoxin production was observed in the analyzed fungi, although their mycotoxin-producing ability has been reported in previous research, which hinders the assessment of bacteria in mycotoxigenic activities. Therefore, further tests are required in this area. Several studies support the effects observed here, where Bacillus and Streptomyces species exhibit antifungal and antimycotoxigenic properties, highlighting the biotechnological potential of the strains studied [41,42,43,44]. Among the evaluated bacteria, the most efficient were B. amyloliquefaciens plantarum MLB3 and B. velezensis CL197, which belong to closely related phylogenetic groups and have a significant history of antifungal use. They employ various mechanisms, including the production of antifungal lipopeptides and volatile organic compounds, as well as competition for nutrients. Furthermore, these bacteria are generally safe for use, posing minimal risks to both animals and humans and having minimal environmental impact [45].

The assessment of metabolite production revealed a wide array of compounds produced by B. velezensis, aligning with the literature, which highlights these as some of its primary mechanisms of action [45]. Although the literature also cites the high potential for antifungal metabolite production by B. amyloliquefaciens [45], only two compounds were detected. In the case of S. griseus CECT 3276, eight compounds were potentially identified; however, their confirmation is challenging due to the broad range of compounds produced by bacteria in this genus. This exacerbates the information available in the databases, making it difficult to conduct a detailed analysis using the techniques employed in this study. Further research specifically for this strain is required to complement the results obtained here. No compounds were detected to B. subtilis MLB2. However, since the metabolites exhibited antifungal activity in the subsequent tests, further research is necessary to determine the compounds produced. Among the compounds produced by B. velezensis CL197 and B. amyloliquefaciens plantarum MLB3 and identified in this study, lipopeptides stand out. This class of compounds, according to the available literature, is proven to be essential for bacteria to exert their antifungal and antimycotoxigenic activity. For instance, Chakraborty et al [43] demonstrated that lipopeptides from Bacillus have the potential to inhibit mycelial growth, conidiogenesis, and conidial germination. This effect was confirmed in this study, where the metabolites produced by the bacteria were able to inhibit fungal growth in both liquid and solid culture media.

To conclude this research, the potential of bacteria to degrade ZEA in simulated digestion was evaluated. In this analysis, the effects observed in liquid culture media were potentiated, with mainly complete degradation of the toxin by the end of the in vitro process and the production of metabolite products in the form of conjugates. It is worth noting that in the in vitro process, there was no reversion of the conjugates to their original form, but further research is needed to assess whether this will also be the observed result in vivo. The bacteria have demonstrated high effectiveness in fungal and mycotoxin control, with broad potential for use in the development of biotechnological products for animal production.

5. Conclusions

The evaluated bacteria demonstrated significant antifungal and antimycotoxin activities with diverse potential applications, such as preventing fungal contamination in stored materials, degrading mycotoxins present in animal feed due to raw material contamination, preventing the absorption of toxins ingested by animals, and more. For a comprehensive characterization of the strains used in this study, further research is required to evaluate the metabolites produced, particularly by B. subtilis MLB2 and S. griseus CECT 3276, and the metabolization products of ZEA generated by the four bioprotective bacteria.

Another unexplored possibility in this study is the establishment of combinations that complement each other and enhance the observed effects. Thus, the research field remains wide open, with significant potential for the development of a biotechnological product for application in animal production. This product could help maintain herd health, food quality and safety, and posing no risks to consumers, contributing to public health maintenance.