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

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23 September 2024

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24 September 2024

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Abstract
The global meat industry has grown substantially, producing 357.39 million tons in 2021, with poultry and pork comprising nearly 73% of this total. However, contamination by mycotoxins, such as zearalenone (ZEA) and fumonisin B1 (FB1), presents a major issue, as these toxins resist common preservation methods. This study explores the potential of bioprotective microorganisms in mycotoxin degradation and fungal control within the animal food production chain, a sector facing significant challenges due to fungal contamination. In this study, 23 bacterial and yeast strains were tested for their ability to degrade ZEA and FB1, and to inhibit mycotoxigenic fungi. Four bacterial strains were highly effective in degrading ZEA: Bacillus amyloliquefaciens MLB3, Bacillus subtilis MLB2, Bacillus velezensis CL197, and Streptomyces griseus CECT 3276. However, no strain achieved satisfactory FB1 degradation. The strains also displayed antifungal activity, inhibiting up to 100% of fungi growth in solid media co-culture tests. Simulated swine and poultry digestion demonstrated complete ZEA degradation after 2 hours of incubation. Metabolite analysis revealed that low-toxicity conjugates were formed. These findings suggest that these bacteria hold significant promise for biotechnological applications in animal production, helping to reduce mycotoxin contamination, improve food safety, and protect both animal and human health.
Keywords: 
Subject: Biology and Life Sciences  -   Animal Science, Veterinary Science and Zoology

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.

Supplementary Materials

The following supporting information can be downloaded at the website of this paper posted on Preprints.org.

Author Contributions

A.G.E.: Conceptualization, Formal analysis, Investigation, Writing - Original Draft. T.dM.N.: Conceptualization, Methodology, Formal analysis, Investigation, Supervision, Writing - Review & Editing. C.L.: Conceptualization, Methodology, Supervision, Writing - Review & Editing. V.D. and A.M.: Methodology. G.M. and F.B.L.: Conceptualization, Methodology, Supervision, Resources, Project administration, Writing - Review & Editing.

Data Availability Statement

All data supporting the findings of this study are available within the paper and its Supplementary Information.

Acknowledgments and Funding

This research was funded by the National Council for Scientific and Technological Development (CNPq), Brazil, grant numbers 142196/2019-3 and 308598/2020-2; by the Coordination of Superior Level Staff Improvement (CAPES), Brazil, grant number 88881.689893/2022-01; by the Spanish Ministry of Science and Innovation (MCIN), Spain, grant number PID2019-108070RB-100; and by the pre-Ph.D. program of the University of Valencia (Atracció de Talent UV-INV-PREDOC19F1-1006684).

Conflicts of Interest

The authors declare no conflicts of interest.

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Table 1. Microorganisms used in this study, sourced from diverse microbiological collections.
Table 1. Microorganisms used in this study, sourced from diverse microbiological collections.
Microorganism Identification Source Original isolation
Bacteria and yeasts
Bacillus amyloliquefaciens CECT 493 Colección Española de Cultivos Tipo Bacterial amylase HT concentrate, United States
Bacillus amyloliquefaciens plantarum MLB3 BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Bacillus licheniformis CECT 20 Colección Española de Cultivos Tipo Unknown
Bacillus megaterium CECT 44 Colección Española de Cultivos Tipo Unknown
Bacillus subtilis MLB2 BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Bacillus thuringiensis CECT 197 Colección Española de Cultivos Tipo Mediterranean flour moth, unknown country
Bacillus velezensis CL197 AgriFood Research and Innovation Laboratory, Pontifícia Universidade Católica do Paraná, Brazil Soil, Brazil
Candida sake CECT 1044 Colección Española de Cultivos Tipo Lambic beer, Belgium
Levilactobacillus (Lactobacillus) brevis BN3 BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Limosilactobacillus (Lactobacillus) fermentum 20_PG2_BHI ZZMK BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Lacticaseibacillus (Lactobacillus) paracasei DSM 2649 German Collection of Microorganisms and Cell Cultures GmbH Silage, unknown country
Lactiplantibacillus (Lactobacillus) plantarum DSM 1055 German Collection of Microorganisms and Cell Cultures GmbH Bread dough, United States
Lacticaseibacillus (Lactobacillus) rhamnosus DSM 20711 German Collection of Microorganisms and Cell Cultures GmbH Unknown
Liquorilactobacillus (Lactobacillus) satsumensis DSM 16230 German Collection of Microorganisms and Cell Cultures GmbH Shochu mash, Japan
Metschnikowia pulcherrima CECT 1691 Colección Española de Cultivos Tipo Fruit of Phoenix dactylifera, Egypt
Paenibacillus chibensis CECT 375 Colección Española de Cultivos Tipo Unknown
Paenibacillus polymyxa CECT 153 Colección Española de Cultivos Tipo Water, unknown country
Pediococcus acidilactici 146 RLT BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Pseudomonas putida MCA BioTech AgriFood Laboratory, Universitat de València, Spain Unknown
Pseudomonas syringae CECT 312 Colección Española de Cultivos Tipo Nicotiana tabacum, Hungary
Saccharomyces cerevisiae DSM 70868 German Collection of Microorganisms and Cell Cultures GmbH African palm wine, unknown country
Streptomyces calvus CECT 3271 Colección Española de Cultivos Tipo Soil, India
Streptomyces griseus CECT 3276 Colección Española de Cultivos Tipo Soil, United States
Filamentous fungi
Aspergillus steiiny CECT 20510 Colección Española de Cultivos Tipo Pollen of bee, Spain
Fusarium graminearum ITEM 126 ITEM Microbial Culture Collection of ISPA Triticum durum kernel, Italy
Fusarium graminearum CECT 20924 Colección Española de Cultivos Tipo Rice carypses, Spain
Fusarium langsethiae ITEM 11031 ITEM Microbial Culture Collection of ISPA Unknown
Fusarium oxysporum CECT 2719 Colección Española de Cultivos Tipo Unknown
Fusarium oxysporum ISPA 7067 ITEM Microbial Culture Collection of ISPA Stalk of rice, Italy
Fusarium sporotrichioides¹ CECT 20165 Colección Española de Cultivos Tipo Emetic material, unknown country
Fusarium verticillioides ITEM 12043 ITEM Microbial Culture Collection of ISPA Unknown
Fusarium verticillioides ISPA 12044 ITEM Microbial Culture Collection of ISPA Unknown
Fusarium verticillioides ITEM 12052 ITEM Microbial Culture Collection of ISPA Unknown
Gibberella zeae CECT 20492 Colección Española de Cultivos Tipo Wheat, Spain
Gibberella zeae CECT 2150 Colección Española de Cultivos Tipo Grain of Zea mays, United States
Penicillium verrucosum VTT D-01847 VTT Culture Collection, VTT Technical Research Centre of Finland Grain, Finland
¹Cataloged as Fusarium poae until December 2021.
Table 2. Screening for the degradation potential (%) of zearalenone (ZEA) and fumonisin B1 (FB1).
Table 2. Screening for the degradation potential (%) of zearalenone (ZEA) and fumonisin B1 (FB1).
Microorganism ZEA degradation FB1 degradation
Bacillus amyloliquefaciens CECT 493 0.00 21.10
Bacillus amyloliquefaciens plantarum MLB3 93.09 28.92
Bacillus licheniformis CECT 20 0.00 20.66
Bacillus megaterium CECT 44 40.65 18.71
Bacillus subtilis MLB2 96.75 11.63
Bacillus thuringiensis CECT 197 6.74 24.94
Bacillus velezensis CL197 94.27 30.03
Candida sake CECT 1044 38.97 14.84
Levilactobacillus (Lactobacillus) brevis BN3 12.91 17.97
Limosilactobacillus (Lactobacillus) fermentum 20_PG2_BHI ZZMK 22.42 18.95
Lacticaseibacillus (Lactobacillus) paracasei DSM 2649 47.02 26.92
Lactiplantibacillus (Lactobacillus) plantarum DSM 1055 38.44 30.49
Lacticaseibacillus (Lactobacillus) rhamnosus DSM 20711 17.61 32.21
Liquorilactobacillus (Lactobacillus) satsumensis DSM 16230 4.45 36.45
Metschnikowia pulcherrima CECT 1691 36.20 18.22
Paenibacillus chibensis CECT 375 2.37 25.68
Paenibacillus polymyxa CECT 153 0.00 25.11
Pediococcus acidilactici 146 RLT 29.25 37.13
Pseudomonas putida MCA 42.55 0.33
Pseudomonas syringae CECT 312 48.41 30.45
Saccharomyces cerevisiae DSM 70868 42.15 24.11
Streptomyces calvus CECT 3271 53.30 24.81
Streptomyces griseus CECT 3276 94.07 14.66
Table 3. Degradation of zearalenone (ZEA) under different conditions and recovery of the toxin through bacterial adsorption (%).
Table 3. Degradation of zearalenone (ZEA) under different conditions and recovery of the toxin through bacterial adsorption (%).
Bacteria T1 T2 T3 T4 T5
Bacillus amyloliquefaciens plantarum MLB3 85.17±4.67 37.63±4.40 92.29±0.50 41.54±7.00 18.59±4.80
Bacillus subtilis MLB2 88.93±4.97 69.58±5.78 92.93±2.17 47.55±6.89 10.60±2.07
Bacillus velezensis CL197 83.92±1.83 60.04±1.64 91.61±5.17 44.39±1.61 10.10±0.68
Streptomyces griseus CECT 3276 84.71±2.98 24.82±6.16 89.34±0.72 11.01±4.08 12.72±3.30
T1: 104 CFU/mL + 1 µg/mL of ZEA, 48h at 37 ⁰C; T2: Cell-free supernatants + 1 µg/mL of ZEA, 48h at 37 ⁰C; T3: Intracellular metabolites + 1 µg/mL of ZEA, 48h at 37 ⁰C; T4: Autoclaved culture + 1 µg/mL of ZEA, 48h at 37 ⁰C; T5: Recovery of ZEA from bacterial biomass adsorption after incubation for 48h at 37 ⁰C in the presence of 1 µg/mL of ZEA.
Table 4. Metabolites of zearalenone (ZEA) produced by the inoculation of Bacillus and S. griseus at a concentration of 104 CFU/mL + 1 µg/mL of ZEA, with incubation for 48 hours at 37 ºC.
Table 4. Metabolites of zearalenone (ZEA) produced by the inoculation of Bacillus and S. griseus at a concentration of 104 CFU/mL + 1 µg/mL of ZEA, with incubation for 48 hours at 37 ºC.
Molecular formula m/z Presumptive compound Concentration* Ref.
Bacillus amyloliquefaciens plantarum MLB3
C24 H34 O11 557.2210 hydroxy-zearalenol-Glc 98.92±0.28 [18]
C24 H32 O11 495.1890 hydroxy-zearalenone-Glc 94.07±0.28 [18]
C30 H44 O15 689.2625 zearalenol-di-Glc 26.54±0.30 [18]
C26 H36 O11 523.2167 zearalenol-Glc-Ac 29.46±0.81 [19]
C24 H32 O10 525.1971 zearalenone-4-beta-D-glucopyranoside 14.69±0.86 [20]
C36 H52 O20 863.3136 zearalenone-tri-Glc 99.10±0.93 [18]
Bacillus subtilis MLB2
C18 H20 O6 331.1174 hydroxy-dehydro-zearalenone 38.48±0.21 [18]
C32 H46 O16 745.2972 zearalenol-di-Glc-Ac 72.70±0.80 [19]
C26 H36 O11 523.2182 zearalenol-Glc-Ac 25.35±0.66 [19]
C36 H52 O20 849.3037 zearalenone-tri-Glc 26.71±0.79 [18]
C18 H26 O5 381.1930 zeranol 15.36±0.51 [21]
C18 H24 O8 S 445.1199 α- or β-zearalenol-Sulf 16.94±0.06 [18]
Bacillus velezensis CL197
C17 H24 O4 291.1597 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 15.50±0.91 [22]
C24 H34 O11 557.2226 hydroxy-zearalenol-Glc 86.98±0.52 [18]
C26 H36 O11 523.2164 zearalenol-Glc-Ac 45.66±0.41 [19]
C24 H32 O10 525.1981 zearalenone-4-beta-D-glucopyranoside 11.20±0.87 [20]
C36 H52 O20 849.3033 zearalenone-tri-Glc 15.49±0.87 [18]
Streptomyces griseus CECT 3276
C18 H20 O6 331.1174 hydroxy-dehydro-zearalenone 68.94±0.28 [18]
C24 H34 O11 557.2241 hydroxy-zearalenol-Glc 88.45±0.74 [18]
C32 H46 O16 731.2736 zearalenol-di-Glc-Ac 62.86±0.55 [19]
C24 H32 O10 525.1980 zearalenone-4-beta-D-glucopyranoside 16.73±0.12 [20]
C27 H34 O13 611.2001 zearalenone-Mal-Glc 59.44±0.02 [23]
C18 H26 O5 321.1704 Zeranol 27.50±0.95 [21]
C18 H24 O8 S 399.1137 α- or β-zearalenol-Sulf 17.84±0.96 [18]
Glc: glucoside; Ac: acetyl; Mal: malonyl; Sulf: sulfate. *Equivalent concentration in ng/mL of ZEA.
Table 5. Inhibition of fungal growth (mm) when in co-culture with the selected bacteria.
Table 5. Inhibition of fungal growth (mm) when in co-culture with the selected bacteria.
Microrganism Control Treatment
Aspergillus steiiny CECT 20510
Bacillus amyloliquefaciens plantarum MLB3 34.5±0.26 21.5±0.62*
Bacillus subtilis MLB2 31.0±0.14
Bacillus velezensis CL197 28.5±0.23*
Streptomyces griseus CECT 3276 34.0±0.44
Fusarium graminearum ITEM 126
Bacillus amyloliquefaciens plantarum MLB3 26.5±0.71 16.5±0.23*
Bacillus subtilis MLB2 26.5±0.15
Bacillus velezensis CL197 23.5±0.71
Streptomyces griseus CECT 3276 27.0±0.66
Fusarium langsethiae ITEM 11031
Bacillus amyloliquefaciens plantarum MLB3 36.5±0.71 23.5±0.32*
Bacillus subtilis MLB2 21.5±0.18*
Bacillus velezensis CL197 19.5±2.12*
Streptomyces griseus CECT 3276 35.5±0.49
Fusarium oxysporum ITEM 2719
Bacillus amyloliquefaciens plantarum MLB3 23.5±0.71 13.5±0.29*
Bacillus subtilis MLB2 21.5±0.73
Bacillus velezensis CL197 19.5±0.71*
Streptomyces griseus CECT 3276 23.5±0.51
Fusarium oxysporum ITEM 7067
Bacillus amyloliquefaciens plantarum MLB3 27.5±0.71 20.5±0.80*
Bacillus subtilis MLB2 22.0±0.27*
Bacillus velezensis CL197 23.0±1.41*
Streptomyces griseus CECT 3276 26.5±0.23
Fusarium verticillioides CECT 12043
Bacillus amyloliquefaciens plantarum MLB3 29.0±2.83 18.5±0.58*
Bacillus subtilis MLB2 27.5±0.51
Bacillus velezensis CL197 25.5±0.71
Streptomyces griseus CECT 3276 28.0±0.50
Fusarium verticillioides CECT 12044
Bacillus amyloliquefaciens plantarum MLB3 42.5±2.12 23.0±0.79*
Bacillus subtilis MLB2 42.5±0.28
Bacillus velezensis CL197 25.5±0.71*
Streptomyces griseus CECT 3276 43.0±0.38
Fusarium verticillioides CECT 12052
Bacillus amyloliquefaciens plantarum MLB3 36.0±1.41 24.5±0.61*
Bacillus subtilis MLB2 32.0±0.64
Bacillus velezensis CL197 22.5±0.71*
Streptomyces griseus CECT 3276 31.5±0.24*
Gibberella zeae CECT 2150
Bacillus amyloliquefaciens plantarum MLB3 42.0±1.41 34.5±0.38*
Bacillus subtilis MLB2 40.5±0.47
Bacillus velezensis CL197 28.5±4.95*
Streptomyces griseus CECT 3276 39.5±0.88
Fusarium graminearum CECT 20924
Bacillus amyloliquefaciens plantarum MLB3 34.5±10.61 23.5±0.71*
Bacillus subtilis MLB2 34.0±0.59
Bacillus velezensis CL197 23.0±5.66*
Streptomyces griseus CECT 3276 33.5±0.63
Fusarium sporotrichioides CECT 20165
Bacillus amyloliquefaciens plantarum MLB3 34.5±0.71 15.5±0.31*
Bacillus subtilis MLB2 33.5±0.78
Bacillus velezensis CL197 24.5±0.71*
Streptomyces griseus CECT 3276 34.0±0.77
Gibberella zeae CECT 20492
Bacillus amyloliquefaciens plantarum MLB3 41.5±9.19 24.0±0.53*
Bacillus subtilis MLB2 42.0±0.49
Bacillus velezensis CL197 25.5±0.71*
Streptomyces griseus CECT 3276 41.0±0.49
Penicillium verrucosum VTT D-01847
Bacillus amyloliquefaciens plantarum MLB3 18.0±0.12 11.5±0.38*
Bacillus subtilis MLB2 15.5±0.79
Bacillus velezensis CL197 13.5±0.73*
Streptomyces griseus CECT 3276 17.0±0.78
*Significant difference (p<0.05) compared to the control group.
Table 6. Antifungal metabolites produced by Bacillus velezensis CL197 and Bacillus amyloliquefaciens plantarum MLB3 (mg/L).
Table 6. Antifungal metabolites produced by Bacillus velezensis CL197 and Bacillus amyloliquefaciens plantarum MLB3 (mg/L).
Incubation Metabolite
Fengycin C Gageostatin B Gageostatin C Marihysin A Plipastatin A2 Plipastatin B1
Bacillus amyloliquefaciens plantarum MLB3
24h nd nd nda nda nd nd
48h nd nd 0.55±0.03b nda nd nd
72h nd nd 1.11±0.07c 2.44±0.19b nd nd
Bacillus velezensis CL197
24h 17.49±2.52a 2.55±0.24a 16.48±1.49a 1.34±0.10a 3.98±0.47a 1.33±0.21a
48h 7.88±0.94b 3.05±0.33b 12.01±1.59b 2.24±0.07b 2.41±0.04b 0.55±0.16b
72h 5.12±0.12c 2.12±0.19a 5.71±0.57c 2.97±0.33c 2.19±0.44b 0.40±0.03b
Different letters within the same column in the same group represent significant differences (p < 0.05). nd: not detected.
Table 7. Antifungal metabolites potenttialy produced by Streptomyces griseus CECT 3276.
Table 7. Antifungal metabolites potenttialy produced by Streptomyces griseus CECT 3276.
Molecular formula Presumptive compound Ref.
C16 H23 N O4 Streptimidone [24]
C29 H36 N2 O6 Frontalamide B [25]
C29 H36 N2 O7 Frontalamide A [25]
C33 H52 O10 Thailandin B [26]
C35 H58 O12 Fungichromin [27]
C36 H58 O10 Faeriefungin [28]
C39 H61 N O14 Rimocidin [29]
C47 H75 N O17 Nystatin A1 [30]
Table 8. Minimum inhibitory and fungicidal concentrations (g/L) of lyophilized metabolites from bioprotective bacteria against different mycotoxigenic fungal strains.
Table 8. Minimum inhibitory and fungicidal concentrations (g/L) of lyophilized metabolites from bioprotective bacteria against different mycotoxigenic fungal strains.
Microrganism Bacillus amyloliquefaciens
plantarum MLB3		 Bacillus subtilis
MLB2		 Bacillus velezensis
CL197		 Streptomyces griseus
CECT 3276
MIC MBC MIC MBC MIC MBC MIC MBC
24h-incubation
Aspergillus steiiny CECT 20510 >500 >500 >500 >500 500 >500 >500 >500
Fusarium graminearum ITEM 126 >500 >500 >500 >500 250 >500 >500 >500
Fusarium langsethiae ITEM 11031 >500 >500 >500 >500 500 >500 >500 >500
Fusarium oxysporum ITEM 2719 >500 >500 >500 >500 500 >500 >500 >500
Fusarium oxysporum ITEM 7067 >500 >500 >500 >500 500 >500 >500 >500
Fusarium verticillioides CECT 12043 >500 >500 >500 >500 250 >500 >500 >500
Fusarium verticillioides CECT 12044 >500 >500 >500 >500 500 >500 >500 >500
Fusarium verticillioides CECT 12052 >500 >500 >500 >500 500 >500 >500 >500
Gibberella zeae CECT 2150 >500 >500 >500 >500 500 >500 >500 >500
Fusarium graminearum CECT 20924 >500 >500 >500 >500 500 >500 >500 >500
Fusarium sporotrichioides CECT 20165 >500 >500 >500 >500 500 >500 >500 >500
Gibberella zeae CECT 20492 >500 >500 >500 >500 500 >500 >500 >500
Penicillium verrucosum VTT D-01847 >500 >500 >500 >500 500 >500 >500 >500
48h-incubation
Aspergillus steiiny CECT 20510 500 500 500 500 500 500 500 500
Fusarium graminearum ITEM 126 500 500 500 500 250 500 500 500
Fusarium langsethiae ITEM 11031 500 500 500 500 500 500 500 500
Fusarium oxysporum ITEM 2719 500 500 500 500 500 500 500 500
Fusarium oxysporum ITEM 7067 500 500 500 500 500 500 500 500
Fusarium verticillioides CECT 12043 500 500 500 500 250 500 500 500
Fusarium verticillioides CECT 12044 500 500 500 500 500 500 500 500
Fusarium verticillioides CECT 12052 500 500 500 500 500 500 500 500
Gibberella zeae CECT 2150 500 500 500 500 250 500 500 500
Fusarium graminearum CECT 20924 500 500 500 500 500 500 500 500
Fusarium sporotrichioides CECT 20165 500 500 500 500 500 500 500 500
Gibberella zeae CECT 20492 500 500 500 500 500 500 500 500
Penicillium verrucosum VTT D-01847 500 500 500 500 500 500 500 500
72h-incubation
Aspergillus steiiny CECT 20510 500 500 500 500 500 500 250 500
Fusarium graminearum ITEM 126 500 500 500 500 250 500 500 500
Fusarium langsethiae ITEM 11031 500 500 500 500 250 500 500 500
Fusarium oxysporum ITEM 2719 500 500 500 500 250 500 500 500
Fusarium oxysporum ITEM 7067 500 500 250 500 500 500 500 500
Fusarium verticillioides CECT 12043 500 500 500 500 250 500 250 500
Fusarium verticillioides CECT 12044 500 500 250 500 500 500 500 500
Fusarium verticillioides CECT 12052 500 500 500 500 500 500 500 500
Gibberella zeae CECT 2150 500 500 500 500 250 500 250 500
Fusarium graminearum CECT 20924 500 500 500 500 500 500 250 500
Fusarium sporotrichioides CECT 20165 250 500 500 500 250 500 500 500
Gibberella zeae CECT 20492 500 500 500 500 250 500 500 500
Penicillium verrucosum VTT D-01847 500 500 500 500 500 500 500 500
Table 9. Inhibition halo of fungal growth (mm) when exposed to lyophilized metabolites generated after 72h of incubation of bioprotective bacteria, at a concentration of 500 g/L.
Table 9. Inhibition halo of fungal growth (mm) when exposed to lyophilized metabolites generated after 72h of incubation of bioprotective bacteria, at a concentration of 500 g/L.
Microrganism Bacillus amyloliquefaciens
plantarum MLB3		 Bacillus subtilis MLB2 Bacillus velezensis CL197 Streptomyces griseus CECT 3276
Aspergillus steiiny CECT 20510 3.9±0.3 nd 5.9±0.8 5.2±0.2
Fusarium graminearum ITEM 126 3.7±0.3 nd 5.6±0.9 5.9±0.1
Fusarium langsethiae ITEM 11031 3.2±0.9 1.3±0.3 5.5±0.1 5.6±0.4
Fusarium oxysporum ITEM 2719 3.5±0.8 1.4±0.3 5.5±0.1 5.6±0.6
Fusarium oxysporum ITEM 7067 3.4±0.8 1.1±0.2 6.1±0.5 5.5±0.6
Fusarium verticillioides CECT 12043 4.0±0.2 nd 6.0±0.1 5.5±0.5
Fusarium verticillioides CECT 12044 3.9±0.3 1.1±0.3 6.2±0.2 5.8±0.1
Fusarium verticillioides CECT 12052 3.4±0.1 nd 6.0±0.9 5.5±0.5
Gibberella zeae CECT 2150 3.5±0.5 1.2±0.2 5.9±0.8 5.3±0.2
Fusarium graminearum CECT 20924 3.7±0.5 nd 5.6±0.1 6.2±0.4
Fusarium sporotrichioides CECT 20165 3.4±0.1 1.1±0.1 5.8±0.3 5.8±0.9
Gibberella zeae CECT 20492 3.5±0.4 nd 5.6±0.3 6.2±0.6
Penicillium verrucosum VTT D-01847 3.3±0.5 nd 5.5±0.6 5.7±0.1
Table 10. Degradation of zearalenone (%) in simulated swine and poultry digestion.
Table 10. Degradation of zearalenone (%) in simulated swine and poultry digestion.
Bacteria Swine simulation Poultry simulation
2h 6h 2h 4h20min
Bacillus amyloliquefaciens plantarum MLB3 80.14±5.71a 100a 97.12±4.36a 100a
Bacillus subtilis MLB2 100b 100a 100a 100a
Bacillus velezensis CL197 60.41±7.23c 96.74±2.13a 100a 100a
Streptomyces griseus CECT 3276 76.18±2.28a 100a 92.44±6.68b 100a
Different letters within the same column represent significant differences (p < 0.05).
Table 11. Presumptive metabolites of zearalenone obtained in poultry digestion simulation supplemented with different bioprotective bacteria.
Table 11. Presumptive metabolites of zearalenone obtained in poultry digestion simulation supplemented with different bioprotective bacteria.
Molecular formula m/z Presumptive compound Concentration* Ref.
Bacillus amyloliquefaciens plantarum MLB3
2h C32 H46 O16 731.2735 zearalenol-di-Glc-Ac 33.09±0.73 [19]
C18 H24 O5 379.1760 α or β-zearalenol 17.10±0.46 [31]
C24 H34 O11 543.2046 hydroxy-zearalenol-Glc 46.20±0.14 [18]
C24 H34 O10 541.2286 zearalenol-Glc 36.51±0.11 [32]
C24 H32 O10 525.1960 zearalenone-4-beta-D-glucopyranoside 33.44±0.71 [20]
C30 H42 O15 641.2442 zearalenone-di-Glc 9.09±0.67 [33]
4h20min C26 H34 O11 567.2059 Ac-zearalenone-Glc or zearalenone-Ac-Glc 42.85±0.85 [19]
C18 H20 O6 377.1243 hydroxy-dehydro-zearalenone 8.11±0.56 [18]
C24 H34 O11 543.2085 hydroxy-zearalenol-Glc 51.29±0.07 [18]
C24 H32 O11 495.1883 hydroxy-zearalenone-Glc 54.74±0.38 [18]
C24 H32 O10 539.2158 zearalenone-4-beta-D-glucopyranoside 19.44±0.66 [20]
Bacillus subtilis MLB2
2h C17 H24 O4 291.1604 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 3.67±0.25 [22]
C24 H32 O10 525.1978 zearalenone-4-beta-D-glucopyranoside 4.69±0.85 [20]
C24 H34 O11 557.2230 hydroxy-zearalenol-Glc 8.49±0.66 [18]
C32 H46 O16 731.2748 zearalenol-di-Glc-Ac 28.61±0.41 [19]
C29 H40 O14 611.2341 zearalenone-Hex-Pen 7.17±0.25 [18]
4h20min C26 H34 O11 567.2056 Ac-zearalenone-Glc or zearalenone-Ac-Glc 37.85±0.22 [19]
C17 H24 O4 291.1600 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 8.60±0.83 [22]
C24 H32 O10 525.1968 zearalenone-4-beta-D-glucopyranoside 15.08±0.16 [20]
C24 H32 O11 555.2093 hydroxy-zearalenone-Glc 15.04±0.24 [18]
C32 H46 O16 745.2958 zearalenol-di-Glc-Ac 15.09±0.30 [19]
C39 H56 O23 891.3163 zearalenol-di-Mal-tri-Glc 16.41±0.26 [18]
C27 H34 O13 625.2141 zearalenone-Mal-Glc 17.44±0.95 [23]
Bacillus velezensis CL197
2h C17 H24 O4 291.1595 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 3.12±0.42 [22]
C24 H32 O10 539.2128 zearalenone-4-beta-D-glucopyranoside 10.84±0.34 [20]
C32 H46 O16 745.2892 zearalenol-di-Glc-Ac 12.37±0.20 [19]
C24 H34 O10 481.2057 zearalenol-Glc 61.40±0.09 [32]
C29 H40 O14 657.2419 zearalenone-Hex-Pen 28.40±0.72 [18]
C18 H24 O5 319.1550 α or β-zearalenol 8.03±0.54 [31]
4h20min C17 H24 O4 291.1593 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 4.15±0.31 [22]
C24 H32 O10 539.2149 zearalenone-4-beta-D-glucopyranoside 24.57±0.39 [20]
C26 H34 O11 567.2057 Ac-zearalenone-Glc or zearalenone-Ac-Glc 56.51±0.32 [19]
C32 H46 O16 731.2767 zearalenol-di-Glc-Ac 42.54±0.71 [19]
C24 H34 O10 527.2133 zearalenol-Glc 7.03±0.93 [32]
C29 H40 O14 657.2428 zearalenone-Hex-Pen 36.99±0.54 [18]
C33 H44 O18 773.2529 zearalenone-Mal-di-Glc 4.28±0.47 [18]
Streptomyces griseus CECT 3276
2h C18 H20 O6 377.1233 hydroxy-dehydro-zearalenone 12.27±0.88 [18]
C18 H24 O6 395.1701 hydroxy-zearalenol 2.84±0.85 [18]
C30 H44 O15 689.2652 zearalenol-di-Glc 2.17±0.84 [18]
C36 H48 O21 815.2596 zearalenol-di-Mal-di-Glc 26.74±0.47 [18]
C24 H32 O10 525.1964 zearalenone-4-beta-D-glucopyranoside 12.57±0.56 [20]
C18 H22 O8 S 397.0954 zearalenone-4-Sulf 28.16±0.14 [19]
C30 H42 O15 641.2415 zearalenone-di-Glc 14.28±0.56 [33]
C29 H40 O14 611.2298 zearalenone-Hex-Pen 20.73±0.50 [18]
C18 H24 O8 S 399.1114 α- or β-zearalenol-Sulf 6.72±0.55 [18]
C20 H24 O6 405.1547 Ac-zearalenone 60.81±0.14 [19]
C24 H34 O10 527.2149 zearalenol-Glc 10.40±0.69 [32]
C39 H54 O24 965.3121 zearalenone-di-Mal-tri-Glc 68.74±0.44 [18]
C33 H44 O18 727.2451 zearalenone-Mal-di-Glc 49.83±0.55 [18]
4h20min C17 H24 O4 291.1595 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 8.89±0.02 [22]
C20 H24 O6 405.1549 Ac-zearalenone 20.20±0.66 [19]
C36 H48 O21 815.2594 zearalenol-di-Mal-di-Glc 25.86±0.48 [18]
C39 H56 O23 891.3155 zearalenol-di-Mal-tri-Glc 27.40±0.06 [18]
C32 H44 O16 743.2722 Ac-zearalenone-di-Glc or zearalenone-Ac-di-Glc 23.47±0.58 [19]
C18 H20 O6 391.1381 hydroxy-dehydro-zearalenone 20.53±0.33 [18]
C32 H46 O16 731.2730 zearalenol-di-Glc-Ac 30.01±0.80 [19]
C24 H32 O10 539.2151 zearalenone-4-beta-D-glucopyranoside 21.85±0.74 [20]
C30 H42 O15 641.2459 zearalenone-di-Glc 15.80±0.60 [33]
Glc: glucoside; Ac: acetyl; Mal: malonyl; Sulf: sulfate; Hex: hexose; Pen: pentose. *Equivalent concentration in ng/mL of ZEA.
Table 12. Presumptive metabolites of zearalenone obtained in swine digestion simulation supplemented with different bioprotective bacteria.
Table 12. Presumptive metabolites of zearalenone obtained in swine digestion simulation supplemented with different bioprotective bacteria.
Molecular formula m/z Presumptive compound Concentration* Ref.
Bacillus amyloliquefaciens plantarum MLB3
2h C32 H44 O16 729.2679 Ac-zearalenone-di-Glc or zearalenone-Ac-di-Glc 11.52±0.33 [19]
C26 H34 O11 521.1995 Ac-zearalenone-Glc or zearalenone-Ac-Glc 58.87±0.06 [19]
C17 H24 O4 291.1598 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 6.15±0.30 [22]
C20 H24 O6 405.1555 Ac-zearalenone 46.39±0.78 [19]
C24 H32 O11 541.1920 hydroxy-zearalenone-Glc 13.14±0.41 [18]
C24 H34 O10 481.2062 zearalenol-Glc 134.27±0.09 [32]
C24 H32 O10 539.2136 zearalenone-4-beta-D-glucopyranoside 14.01±0.35 [20]
C18 H24 O5 379.1764 α or β-zearalenol 7.17±0.86 [31]
C18 H20 O6 377.1230 hydroxy-dehydro-zearalenone 24.96±0.39 [18]
C24 H34 O11 543.2048 hydroxy-zearalenol-Glc 78.07±0.31 [18]
C36 H48 O21 815.2601 zearalenol-di-Mal-di-Glc 19.23±0.28 [18]
C18 H22 O8 S 397.0954 zearalenone-4-sulfate 17.41±0.72 [34]
C29 H40 O14 657.2431 zearalenone-Hex-Pen 29.32±0.31 [18]
6h C24 H34 O11 497.2016 hydroxy-zearalenol-Glc 75.16±0.74 [18]
C24 H34 O10 527.2127 zearalenol-Glc 99.47±0.54 [32]
C18 H22 O8 S 397.0958 zearalenone-4-sulfate 254.37±0.62 [34]
C30 H42 O15 641.2448 zearalenone-di-Glc 10.33±0.20 [33]
C18 H24 O8 S 399.1117 α- or β-zearalenol-Sulf 72.55±0.01 [18]
C24 H32 O11 495.1862 hydroxy-zearalenone-Glc 44.32±0.83 [18]
C24 H32 O10 479.1944 zearalenone-4-beta-D-glucopyranoside 11.25±0.93 [20]
Bacillus subtilis MLB2
2h C26 H34 O11 521.2000 Ac-zearalenone-Glc or zearalenone-Ac-Glc 17.17±0.58 [19]
C17 H24 O4 291.1604 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 5.04±0.96 [22]
C18 H20 O6 331.1182 hydroxy-dehydro-zearalenone 3.38±0.58 [18]
C24 H32 O11 541.1915 hydroxy-zearalenone-Glc 15.65±0.17 [18]
C26 H36 O11 523.2175 zearalenol-Glc-Ac 74.89±0.49 [19]
C18 H20 O6 331.1191 hydroxy-dehydro-zearalenone 9.34±0.85 [18]
C24 H34 O11 557.2229 hydroxy-zearalenol-Glc 107.05±0.25 [18]
C24 H32 O10 525.1958 zearalenone-4-beta-D-glucopyranoside 24.38±0.04 [20]
C18 H22 O8 S 397.0963 zearalenone-4-sulfate 18.91±0.69 [34]
C29 H40 O14 671.2568 zearalenone-Hex-Pen 38.04±0.25 [18]
6h C32 H44 O16 743.2777 Ac-zearalenone-di-Glc or zearalenone-Ac-di-Glc 5.68±0.45 [19]
C30 H44 O15 689.2665 zearalenol-di-Glc 135.44±0.32 [18]
C33 H44 O18 787.2613 zearalenone-Mal-di-Glc 14.81±0.44 [18]
C18 H20 O5 375.1471 dehydro-zearalenone 6.36±0.92 [35]
C18 H20 O6 331.1186 hydroxy-dehydro-zearalenone 8.75±0.61 [18]
C24 H34 O11 543.2080 hydroxy-zearalenol-Glc 85.54±0.85 [18]
C24 H32 O11 495.1885 hydroxy-zearalenone-Glc 36.37±0.79 [18]
C32 H46 O16 685.2689 zearalenol-di-Glc-Ac 7.92±0.61 [19]
C24 H34 O10 527.2140 zearalenol-Glc 151.77±0.40 [32]
C24 H32 O10 525.1963 zearalenone-4-beta-D-glucopyranoside 24.05±0.61 [20]
C18 H22 O8 S 397.0958 zearalenone-4-sulfate 251.24±0.82 [34]
C30 H42 O15 641.2443 zearalenone-di-Glc 8.73±0.18 [33]
C27 H34 O13 625.2133 zearalenone-Mal-Glc 5.79±0.18 [23]
C18 H24 O8 S 399.1114 α- or β-zearalenol-Sulf 72.85±0.22 [18]
Bacillus velezensis CL197
2h C32 H44 O16 729.2565 Ac-zearalenone-di-Glc or zearalenone-Ac-di-Glc 16.48±0.38 [19]
C17 H24 O4 291.1598 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 5.30±0.47 [22]
C32 H46 O16 731.2733 zearalenol-di-Glc-Ac 75.54±0.81 [19]
C18 H24 O5 379.1762 α or β-zearalenol 11.79±0.38 [31]
C18 H20 O6 391.1378 hydroxy-dehydro-zearalenone 59.25±0.65 [18]
C24 H32 O11 495.1881 hydroxy-zearalenone-Glc 196.25±0.98 [18]
C39 H56 O23 937.3181 zearalenol-di-Mal-tri-Glc 83.98±0.35 [18]
C24 H34 O10 541.2234 zearalenol-Glc 23.63±0.25 [32]
C24 H32 O10 525.1956 zearalenone-4-beta-D-glucopyranoside 19.38±0.34 [20]
C18 H22 O8 S 397.0960 zearalenone-4-sulfate 36.49±0.57 [34]
C29 H40 O14 611.2360 zearalenone-Hex-Pen 75.46±0.42 [18]
C27 H34 O13 565.1914 zearalenone-Mal-Glc 19.45±0.79 [23]
C36 H52 O20 849.3013 zearalenone-tri-Glc 22.83±0.81 [18]
6h C17 H24 O4 291.1594 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 5.79±0.75 [22]
C39 H56 O23 937.3187 zearalenol-di-Mal-tri-Glc 17.54±0.63 [18]
C24 H34 O10 481.2058 zearalenol-Glc 67.68±0.19 [32]
C24 H32 O10 525.1969 zearalenone-4-beta-D-glucopyranoside 24.60±0.80 [20]
C18 H22 O8 S 397.0958 zearalenone-4-sulfate 258.32±0.36 [34]
C29 H40 O14 657.2412 zearalenone-Hex-Pen 17.90±0.43 [18]
C18 H24 O5 379.1757 α or β-zearalenol 26.50±0.50 [31]
C20 H24 O6 405.1573 Ac-zearalenone 5.32±0.86 [19]
C24 H32 O11 541.1921 hydroxy-zearalenone-Glc 27.85±0.57 [18]
C33 H46 O18 789.2807 zearalenol-Mal-di-Glc 10.88±0.19 [36]
C33 H44 O18 773.2571 zearalenone-Mal-di-Glc 8.80±0.88 [18]
C27 H34 O13 625.2141 zearalenone-Mal-Glc 8.81±0.85 [23]
C18 H24 O8 S 399.1116 α- or β-zearalenol-Sulf 77.30±0.21 [18]
Streptomyces griseus CECT 3276
2h C20 H24 O6 405.1542 Ac-zearalenone 26.02±0.31 [19]
C18 H20 O6 391.1390 hydroxy-dehydro-zearalenone 16.55±0.23 [18]
C26 H34 O11 581.2232 Ac-zearalenone-Glc or zearalenone-Ac-Glc 3.39±0.75 [19]
C17 H24 O4 351.1811 1-(3,5-dihydroxyphenyl)-10′-hydroxy-1-undecen-6-one 3.21±0.29 [22]
C18 H24 O6 381.1547 hydroxy-zearalenol 4.70±0.14 [18]
C32 H46 O16 731.2724 zearalenol-di-Glc-Ac 45.64±0.26 [19]
C39 H56 O23 937.3252 zearalenol-di-Mal-tri-Glc 9.16±0.87 [18]
C24 H34 O10 527.2116 zearalenol-Glc 41.92±0.39 [32]
C26 H36 O11 523.2157 zearalenol-Glc-Ac 62.14±0.39 [19]
C24 H32 O10 525.1993 zearalenone-4-beta-D-glucopyranoside 31.71±0.78 [20]
C18 H22 O8 S 397.0956 zearalenone-4-sulfate 23.88±0.92 [34]
C29 H40 O14 611.2330 zearalenone-Hex-Pen 16.59±0.36 [18]
6h C26 H34 O11 521.2059 Ac-zearalenone-Glc or zearalenone-Ac-Glc 11.96±0.30 [19]
C24 H32 O10 525.1964 zearalenone-4-beta-D-glucopyranoside 15.58±0.66 [20]
C39 H54 O24 965.3100 zearalenone-di-Mal-tri-Glc 10.45±0.59 [18]
C20 H24 O6 405.1545 Ac-zearalenone 20.06±0.73 [19]
C24 H34 O10 541.2286 zearalenol-Glc 18.71±0.53 [32]
C18 H22 O8 S 397.0958 zearalenone-4-sulfate 255.97±0.11 [34]
C18 H24 O8 S 399.1117 α- or β-zearalenol-Sulf 75.65±0.94 [18]
Glc: glucoside; Ac: acetyl; Mal: malonyl; Sulf: sulfate; Hex: hexose; Pen: pentose. *Equivalent concentration in ng/mL of ZEA.
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