Probiotic and adsorbent viability of aflatoxin B1 by the yeast Saccharomyces cerevisiae A8L3 isolated from fish farming environment

Viabilidade probiótica e adsorvente de aflatoxina B1 pela levedura Saccharomyces cerevisiae A8L3 isolada de ambiente de piscicultura

Abstract

Due to the increasing demand for sustainable alternatives to antibiotics in aquaculture, the selection of indigenous probiotics with multi-functional properties is essential for improving fish health and food safety. This research aimed to evaluate the probiotic potential and Aflatoxin B1 (AFB1) adsorption capacity of the yeast strain S. cerevisiae A8L3, isolated from a fish farm environment. The isolate, belonging to the NUEPPA culture collection, was subjected to in vitro assays to determine its autoaggregation and co-aggregation abilities, antimicrobial activity against pathogens, and tolerance to simulated stomach (pH 2.0) and intestinal (pH 7.0) conditions of Nile tilapia (Oreochromis niloticus). Results indicated that S. cerevisiae A8L3 possesses robust aggregative properties and effective bacterial inhibition for all strains evaluated. Furthermore, the strain demonstrated high viability and the ability to multiply under simulated gastrointestinal conditions. In addition to these probiotic traits, A8L3 showed a significant capacity for AFB1 adsorption. These findings confirm that S. cerevisiae A8L3 meets the essential requirements for inclusion in fish diets. The strain demonstrates significant potential as a dual-action biological agent, capable of enhancing pathogen resistance and mitigating risks associated with mycotoxin-contaminated feed, thus offering a promising strategy for sustainable Nile tilapia farming.

Keywords:
mycotoxin; biocontrol; aquaculture health; Nile tilapia

Resumo

Devido à crescente demanda por alternativas sustentáveis aos antibióticos na aquicultura, a seleção de probióticos autóctones com propriedades multifuncionais é essencial para melhorar a saúde dos peixes e a segurança alimentar. Esta pesquisa teve como objetivo avaliar o potencial probiótico e a capacidade de adsorção de Aflatoxina B1 (AFB1) da cepa de levedura S. cerevisiae A8L3, isolada de um ambiente de piscicultura. O isolado, pertencente à coleção de culturas do NUEPPA, foi submetido a ensaios in vitro para determinar suas habilidades de autoagregação e coagregação, atividade antimicrobiana contra patógenos e tolerância às condições simuladas de estômago (pH 2.0) e intestino (pH 7.0) da tilápia-do-Nilo (Oreochromis niloticus). Os resultados indicaram que a S. cerevisiae A8L3 possui propriedades agregativas robustas e inibição bacteriana eficaz para todas as cepas avaliadas. Além disso, a cepa demonstrou alta viabilidade e capacidade de multiplicação sob condições gastrointestinais simuladas. Somado a essas características probióticas, a A8L3 apresentou uma capacidade significativa de adsorção de AFB1. Estes achados confirmam que a S. cerevisiae A8L3 atende aos requisitos essenciais para inclusão em dietas de peixes. A cepa demonstra potencial significativo como um agente biológico de dupla ação, capaz de aumentar a resistência a patógenos e mitigar os riscos associados a rações contaminadas por micotoxinas, oferecendo, assim, uma estratégia promissora para a piscicultura sustentável de tilápia-do-Nilo.

Palavras-chave:
micotoxina; biocontrole; sanidade aquícola; tilápia-do-Nilo

1. Introduction

Several types of microorganisms have been studied as probiotics in human and animal nutrition, aiming at improving the responses inherent to the digestive system. In this scenario, probiotics can be defined as live microorganisms capable of promoting intestinal balance and producing positive effects on the health or physiology of individuals that ingest them (Caruffo et al., 2015; Hill et al., 2014; Nogueira and Gonçalves, 2011). The microorganisms studied and used as probiotics include bacteria of the genera Lactobacillus, Bifidobacterium, and Lactococcus, and yeasts such as Saccharomyces boulardii and Saccharomyces cerevisiae (Terhaag et al., 2020).

Similar to bacteria, yeasts belong to other groups of microorganisms that have a series of attributes that can be used as probiotic agents. Yeasts also show several benefits, adsorbing mycotoxins in their cell walls and having several immunostimulating components, e.g., β-glucans, which can favor protective responses against pathogens (Vargas et al., 2021). Other factors in which yeasts have stood out concern the protection of the health of both animals and humans, benefits that have aroused the interest of researchers in the use of these microorganisms to decontaminate mycotoxins produced by mycotoxigenic fungi (Hassan and Zhou, 2018; Pfliegler et al., 2015).

Filamentous fungi are the main producers of mycotoxins, which are considered secondary metabolites with low molecular weight arising from the metabolism of these microorganisms. Most of the metabolites produced have good stability at high temperatures and can resist in cereals and animal food even during the extrusion process, in addition to resisting the most varied pH ranges in the gastrointestinal tract of fish (Campagnollo et al., 2020; Matejova et al., 2017; Pereyra et al., 2018).

When these mycotoxins are present in food, especially in feed storage, they can cause significant economic losses and reduce the production rate of animals. Mycotoxins are highly toxic metabolites that pose high risks and can lead to high mortality rates in fish farming (Matejova et al., 2017; Tibola and Fernandes, 2020).

Several alternatives have been employed to control mycotoxins due to their toxic activity, e.g., prevention of fungal contamination and multiplication, food decontamination, and reduction in the absorption of mycotoxins present in consumed food in the gastrointestinal tract by inhibition or adsorption (Luo et al., 2020).

Among mycotoxin control strategies, biological detoxification has shown the most promising results, being carried out by yeasts with proven probiotic activity. These microorganisms can adsorb mycotoxins, thus eliminating or reducing the levels of toxins in the food to a safe level (Luo et al., 2020; Pereyra et al., 2015; Pinheiro et al., 2017).

From this perspective, the present study aimed to verify whether the yeast strain Saccharomyces cerevisiae A8L3 isolated from fish farms has probiotic and adsorbent capacity for aflatoxin B1 in in vitro tests.

2. Material and Methods

2.1. In vitro probiotic tests

The S. cerevisiae strain A8L3, isolated from a pisciculture environment and belonging to the culture collection of the Microbiological Food Control Laboratory (NUEPPA/CCA/UFPI), was used in this study. Initially, a total of 30 strains were isolated and morphologically identified as yeasts, according to Pitt and Hocking (2009). After a screening process based on low pH tolerance, ten strains were selected for molecular identification, including the A8L3 strain.

Molecular identification was performed using PCR-fingerprinting with the GTG5 primer (5'-GTGGTGGTGGTGGTG-3'). To confirm these results, the ITS region was sequenced using the primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The amplified PCR products were sent to Macrogen Inc. (Seoul, South Korea) for DNA sequencing. The resulting sequences were analyzed using the Basic Local Alignment Search Tool (BLAST) through the NCBI (GenBank) database to determine the species identity.

Subsequently, this strain was evaluated for its probiotic potential through in vitro assays simulating the gastrointestinal physiology of Nile tilapia (O. niloticus)."

To evaluate the probiotic viability capacity, the yeast S. cerevisiae A8L3 was subjected to the following tests: yeast autoaggregation and coaggregation capacity, inhibition of pathogenic microorganisms, and simulated tolerance to stomach pH conditions (pH 2.0) and intestine pH conditions (pH 7.0) of Nile tilapia, in addition to aflatoxin B1 adsorption tests.

2.2. Yeast self-aggregation and co-aggregation capacity

The methodology described by Pizzolitto et al. (2012) was used, with some modifications, to determine the cell-cell adhesion property of yeasts through autoaggregation (yeast strain only) and coaggregation (yeast and pathogen) assays. The resuspended inocula of the yeast strain S. cerevisiae A8L3 were cultivated in YPD broth and incubated at 37 ºC for 24 hours. After incubation, the cells were centrifuged at 5,000 rpm for 10 minutes at room temperature, then washed twice in sterile distilled water, resuspended in 4.0 mL of phosphate buffer saline (PBS - pH 7.2), and homogenized in a vortex shaker. In the two assays, the cell suspensions were standardized with an optical density (DO_initial)_600nm = 0.5. The tubes with the suspensions were incubated at 37 ºC for two hours without stirring. Then, the absorbance values of the top layer were measured for final optical density. (DO_final)_600nm in a spectrophotometer. The autoaggregation percentage of the strain was determined through the following Equation 1:

Autoaggregation (%) =

$\left[1-\left(\frac{D{O}_{final}}{D{O}_{initial}}\right)\right]\times 100$ (1)

Where:

DO_final= Final optical density after two hours of incubation

DO_initial= Initial optical density at time zero of incubation

The method used to prepare yeast suspensions for coaggregation was the same as that used for the autoaggregation assay described by Pizzolitto et al. (2012). Four different pathogenic microorganisms provided by the NUEPPA Reference Microorganism Collection were used in this assay: Escherichia coli INCQS 00033 (ATCC 25922); Salmonella enterica subsp. enterica serovar Typhimurium INCQS 00150 (ATCC 14028; Staphylococcus aureus INCQS 00015 (ATCC 25923), and Aeromonas hydrophila INCQS 00318 (IOC/FDA 110-36), which pose health risks to fish and fish consumers. Each pathogenic bacterium was cultured in brain heart infusion (BHI) and incubated for 24 hours at 37 °C. Then, each culture was centrifuged at 5.000 rpm for 10 minutes. The supernatant was then discarded and the cells were resuspended in PBS (pH 7.2). Dilutions were performed with the same buffer solution to adjust the final concentration of both yeast and bacteria to OD₆₀₀ nm = 0.5. Subsequently, equal volumes (2.0 mL) of yeast and pathogenic bacteria were mixed in sterile tubes, homogenized in a vortex shaker, and incubated at 37 ºC for two hours without shaking. Then, the supernatant of the mixture described above (DO_mix) and the control tubes with suspensions of yeast (DO_yeast) and bacteria (DO_pathogen) had their absorbance measured at 600 nm. The following Equation 2 was applied to determine the percentage of co-aggregation:

Co-aggregation(%) =

$\left[1-D{O}_{Mix}÷\left(\frac{D{O}_{Pathogen}+D{O}_{Yeast}}{2}\right)\right]\times 100$ (2)

Where:

DO_Mix= Optical density of the yeast + pathogen mixture;

DO_Pathogen= Pathogen optical density

DO_yeast= Yeast optical density

The autoaggregation and coaggregation assays were conducted in three independent replicates. The data were subjected to statistical analysis and are presented as mean values ± SD (standard deviation).

2.3. Inhibition of pathogenic microorganisms

The yeast was tested for inhibition of pathogenic microorganisms using the slab test method in YPD medium (Strus, 1998). The strains of pathogenic bacteria used in this assay were the same as those used in the co-aggregation test. In order to adjust the bacterial concentration to reach 0.5 on the McFarland scale, the pathogenic bacteria were sown on nutrient agar at 37.0 ºC for 24 hours in a microbiological incubator. Subsequently, inocula from each culture were removed from the agar with a platinum loop for resuspension in saline solution (0.9%), proceeding in this way until bacterial suspensions corresponding to 0.5 on the McFarland scale were obtained. After adjusting the inoculum, with the aid of a sterilized swab (Cral®), the bacteria were sown in streaks across the entire surface of Petri dishes containing Nutrient agar. Then, 14-mm YPD agar disks containing the yeasts previously sown at 25.0 ºC for 48 hours were aseptically cut and added to the Nutrient agar plates that were previously sown with the pathogenic bacteria. To ensure the accuracy of the measurements and to eliminate any possibility of interference or overlapping between inhibition halos, a single disk was placed in the center of each Petri dish. The assays were performed in duplicate, with each replicate corresponding to an individual plate.

The diameters of the growth inhibition zones around the agar plates were measured after 24 hours of incubation at 37.0 ºC. The results were described in millimeters (mm) by subtracting the diameter of the YPD agar disk.

2.4. Simulated tolerance of low pH conditions and bile salts

The standardization of the inoculum followed a methodology adapted by (Pizzolitto et al., 2012). Briefly, 100 μL of the suspension was added to 900.0 μL of YPD broth adjusted to pH 2.0. The solutions containing the cells were incubated under constant stirring (150 rpm) for different time intervals: 0, 4, 8, and 12 hours at 30 ± 2.0 ºC. The zero-hour (0 h) sampling was performed immediately after inoculation to establish the initial viability. At the end of each incubation time, aliquots of 100.0 μL were removed to count the viable cells by serial decimal dilution and spread-plating on a YPD agar surface. The plates were incubated for 24 h at 37 °C.

In order to determine the viability and tolerance of yeast strains in the presence of bile salts under the conditions of the Nile tilapia intestine, the test was carried out using a methodology similar to the low pH tolerance test. For this, YPD broth supplemented with 0.5% bile salts (Sigma-Aldrich®) adjusted to pH 7.0 was used. At the end of different incubation times (4, 8, and 12 hours), aliquots of 100 μL were taken to count viable cells through serial decimal dilution, followed by spreading on a YPD agar surface. The plates were incubated for 24 hours at 37.0 °C. Assay controls were performed by subjecting each strain to the same procedure described, but with inoculation in YPD broth at pH 7.0 and without the addition of bile salts.

2.5. AFB1 adsorption assay

The AFB1 adsorption test was performed according to Pizzolitto et al. (2011) and Poloni et al. (2015) with some modifications. In order to create the initial AFB1 solution, the material was extracted from a nucleus produced from the standard strain of A. parasiticus NRRL 2999 (USDA, Agricultural Research Service, Peoria, IL), at a concentration of 2.67 μg/mL (Magnoli et al., 2011).

Suspensions were prepared at a concentration of 107 cells/mL from previously sown MEA tubes with isolates of the S. cerevisiae A8L3 strain, and standardization was carried out using a Neubauer chamber. Working solutions of AFB1 (25 and 50 ng/mL) in PBS (pH 2.0 and 7.0) were prepared in microtubes. Next, 1.0 mL of each cell suspension was centrifuged for 15 minutes at 5000 rpm at room temperature. Soon after, they were washed twice with distilled water and centrifuged. Then, 1.0 mL of PBS solution (pH 2.0) containing AFB1 was added to simulate the acidity of the Nile tilapia stomach and incubated at 30.0 ºC for 60 minutes with constant stirring (150 rpm). Next, the tubes were centrifuged and 1.0 mL of PBS at pH 7.0 containing AFB1 at the tested concentrations (25.0 and 50.0 ng/mL) was added to the cell concentrates. Soon after, they were incubated for 60 minutes at 30.0 ± 2 ºC and subjected to constant stirring.

After this period, the cells were pelleted by centrifugation for 15 min at 5.000 rpm at room temperature, and the supernatant containing unbound mycotoxins was collected and stored at -20 ºC for analysis of the percentage of adsorption by high-performance liquid chromatography (CLAE).

The detection and quantification of unadsorbed AFB1 were carried out using a Shimadzu® HPLC chromatograph (model Prominence) with an RF-10AXL Super fluorescence detector, according to the methodology proposed by (Trucksess et al., 1994) and (Scudamore and MacDonald, 1998). Chromatographic separations were performed using a C18 reversed-phase column (silica gel, 150 x 4.6 mm id., 5.0 μm particle size, Varian, Inc. Palo Alto, USA). For analysis, a 200 μL aliquot of the sample was derivatized with 700 μL of a solution consisting of trifluoroacetic acid: glacial acetic acid: water (20:10:70, v/v/v). Acetonitrile: methanol: water (17:17:66 v/v) was used as the mobile phase at a flow rate of 1.5 mL/min. The injection volume was 20.0 μL.

The toxin quantification curve was made by measuring the areas and interpolating them into a calibration curve constructed with different concentrations of AFB1 standard, from which the detection and quantification limits of the technique were extracted. The quantifications of adsorbed AFB1 were established through the correlation between the peak areas of the samples and the standard curve. The adsorption percentages of AFB1 were performed by the following Equation 3:

$$\begin{array}{c}\text{AFB1 adsorption= 100 x}\\ \left[\text{1 -}\left(\begin{array}{l}\text{sample peak area/}\\ \text{mycotoxin peak area}\\ \text{in positive control}\end{array}\right)\right]\end{array}$$ (3)

2.6. Statistical analysis

The data obtained were expressed as mean ± standard deviation (SD). For the evaluation of probiotic potential, data were expressed as percentages (autoaggregation and coaggregation) and in log CFU/mL (tolerance to low pH and bile salts).

For the AFB1 adsorption assays, data were expressed as both the concentration of adsorbed mycotoxin (ng/mL) and the adsorption percentage. Statistical analysis was performed using Analysis of Variance (ANOVA) through the PROC GLM procedure of SAS® University Edition software. Treatment means were compared using Tukey’s test at a significance level of p < 0.05.

3. Results and Discussion

The strain had a percentage of self-aggregation greater than 60%, being considered good due to the score (+) that gives it the ability to adhere to cells of the gastrointestinal tract, in addition to qualities desired for a good probiotic, e.g., better balance of the fish microbiota. The results obtained for the strain S. cerevisiae A8L3 in the autoaggregation tests showed an aggregation score of (62.2% ± 7.89), being considered a strong aggregation, score (+), according to the classification of Kos et al. (2003), values that are in line with those obtained by Pinheiro et al. (2020), who also used S. cerevisiae yeast strains from fish farming and liquor production environments.

The same species isolated from corn-based fermented foods in Nigeria was tested with the same methodology and showed strong aggregation, with values above 80% (Ogunremi et al., 2015), which gives the individual excellent intestinal colonization, as the cell wall of these yeasts is rich in carbohydrates and properties that provide adhesion and enable their fixation and multiplication in the intestinal epithelium.

The in vitro probiotic co-aggregation and antimicrobial activity assays, carried out with the yeast strain S. cerevisiae A8L3, and tolerance, which is based on the physiological status of Tilapia, are presented in Tables 1 and 2. In assays for co-aggregation capacity (Table 1), the results showed that there was binding efficiency with bacterial strains with higher values for S. Typhimurium (43.77%) and lower values for Escherichia coli (27.87%). However, in general, all bacteria showed good coaggregation capacity with the tested strain, which favors protection against pathogenic microorganisms in the body of the host.

Table 1
Probiotic capacity of the S. cerevisiae A8L3 strain for coaggregation and antimicrobial activity for the microorganisms tested.

Bacterial strains tested ( ** )	Coaggregation (%)	Antimicrobial activity Inhibition halo in mm and interpretation scores *
E. coli	27.87c ± 2.25	19.50a ± 0.58 (+++)
S. Typhimurium	43.77a ± 1.45	18.50a ± 1.15 (+++)
S. aureus	37.10b ± 1.50	14.00b ± 0.58 (++)
A. hydrophila	31.70c ± 1.20	18.50a ± 0.58 (+++)

• Ẋ: mean, DP: standard deviation. Means followed by lowercase letters in the same column differ significantly by Tukey’s test at 5% probability (p<0.05).
• ^*
  Antibiogram and its interpretation scores: (-): inhibition zone ≤ 3.0 mm; (+): inhibition zone ≥ 3.0 mm and ≤ 9.0 mm; (++): inhibition zone ≥ 10.0 mm and ≤ 15.0 mm; (+++) inhibition zone > 15 mm;
• (**) Escherichia coli INCQS 00033 (ATCC 25922); Salmonella enterica enterica Typhimurium INCQS 00150 (ATCC 14028; Staphylococcus aureus INCQS 00015 (ATCC 25923) e Aeromonas hydrophila INCQS 00318 (IOC/FDA 110-36).

Table 2
Tolerance to low pH (pH 2.0) and bile salts (pH 7.0) on the viability of S. cerevisiae A8L3 for 12 hours.

pH	Count of S. cerevisiae A8L3 (log CFU/mL) / Time (Hours) (Ẋ ± DP)
	0	4	8	12
2.0	6.83aB ± 0.75	7.19aAB ± 0.16	7.87aA ± 0.09	8.17aA ± 0.08
7.0 (control)	6.67aB ± 0.26	7.54aAB ± 0.08	7.65aAB ± 0.15	8.48aA ± 0.03
7.0 (with bile)	6.89aB ± 0.16	6.93aB ± 0.21	7.63aAB ± 0.03	8.26aA ± 0.01

• Means followed by different lowercase letters in the same column and different uppercase letters in the same row differ significantly by Tukey’s test (p<0.05). Viable cell count (log CFU.mL-1).

With regard to the tests carried out to verify antibacterial activity (Table 1), the yeast S. cerevisiae A8L3 proved to be an excellent inhibitor of the growth of pathogenic microorganisms used in the test, which shows that this strain has antagonistic activity and can be used as a probiotic.

The coaggregation capacity of a probiotic is linked to the prevention of pathogens that can overcome the barrier of the intestinal lumen, causing damage to the host (Abbasiliasi et al., 2017). The S. cerevisiae A8L3 strain showed good results in the co-aggregation test, especially for S. Typhimurium (Table 1), which is a potential enteric pathogen. This capacity can be variable, depending on the strain of S. cerevisae and the pathogenic bacterial species tested (Pinheiro et al., 2020; Poloni et al., 2021). Because it is capable of binding to these intestinal pathogens, the A8L3 strain can be used to prevent illnesses associated with these agents, acting as a barrier to prevent pathogens from invading the gastrointestinal tract.

The S. cerevisiae A8L3 strain, in addition to having the ability to co-aggregate pathogens, also has the ability to inhibit the tested pathogens. The antimicrobial activity observed against the pathogens suggests that S. cerevisiae A8L3 produces inhibitory extracellular compounds, such as organic acids, ethanol, or killer toxins. The zones of inhibition developed independently of direct prior contact between the yeast cells and the pathogens in the initial phase of the assay, indicating that the observed antagonism is mediated by the diffusion of secondary metabolites into the medium. Therefore, in addition to the probiotic capacity already discussed previously, the strain S. cerevisiae A8L3 can contribute to protecting the host's health by avoiding possible developments of resistance to antimicrobials used in fish farming. Furthermore, this strain can be researched in the future as a potential agent for the production of drugs with antibiotic capacity.

Palma et al. (2015), emphasize that the use of these strains with proven probiotic activity can be used as possible prophylactic and therapeutic carriers to combat specific diseases. (Rajkowska et al., 2012) used the same methodology used in the slab test and found inhibition only for Staphylococcus aureus when using the yeast S. cerevisiae var. boulardii, whereas the results of the present study show antagonistic activity for all microorganisms tested.

Table 2 represents the results for the effect of the simulated passage of the yeast S. cerevisiae A8L3 through the pH of the stomach (pH 2.0) and intestine (pH 7.0) and the addition of 0.5% bile on its viability during 12 hours. There was an interaction (p<0.05) between pH levels and the counting time of viable cells. Thus, there was no difference in the count of S. cerevisiae A8L3 (p>0.05) for all pH levels at the four times observed. However, the viable cell count was similar for 8 and 12 hours (p>0.05) and higher than time zero (p<0.05) in both the stomach and intestine (pH = 7.0) with the addition of bile salts.

It is noteworthy that a microorganism with probiotic potential must be able to overcome the barrier of the intestinal lumen with its different pH values throughout the gastrointestinal tract, in addition to facing the physiological and metabolic differences that these regions have. Therefore, it is essential that this microorganism resists the most varied situations (Abbasiliasi et al., 2017; Banerjee and Ray, 2017).

The tested strain of S. cerevisiae A8L3 was able to survive and multiply satisfactorily in simulated in vitro pH conditions of the stomach and intestine of Nile tilapia (Table 2) (Rotta, 2003). The results obtained in this research are in line with other studies that report the ability of yeasts to survive at high concentrations during simulated gastrointestinal conditions (Greppi et al., 2017; Poloni et al., 2017; Zeng et al., 2019). The yeast strain S. cerevisiae A8L3 showed excellent passage at the different times observed and, in addition to passing through the GIT, multiplication occurred under the conditions tested, representing desired qualities for a good probiotic.

These findings are consistent with studies published in the Silva et al. (2015) and Ferreira et al. (2021), which emphasize that the selection of microorganisms capable of resisting the gastrointestinal tract stressors is a prerequisite for effective colonization in O. niloticus.

Table 3 expresses the percentages of AFB1 adsorption in vitro by the strain S. cerevisiae A8L3 isolated in a fish farming environment. The strain tested was capable of adsorbing AFB at the concentrations of 25 ng mL^-1 and 50 ng mL^-1 at rates, with values above 50% adsorption.

Table 3
Adsorption (%) at the concentrations of 25 and 50 ng mL^-1 of AFB1 by the S. cerevisiae A8L3 strain isolated from a fish farming environment.

Concentration AFB1 (ng mL-1)	AFB1 Adsorbed (ng mL-1)	% AFB1 Adsorbed (ng mL-1)
25 (Ẋ ± DP)	13.93 ± 1.67	55.70a ± 5.74
50 (Ẋ ± DP)	25.62 ± 2.70	51.23a ± 5.07

• Ẋ: mean, DP: standard deviation. Means followed by different lowercase letters in the same column differ significantly by Tukey’s test at 5% probability (p<0.05).

The strain S. cerevisiae A8L3 (Table 3) exhibits desirable probiotic characteristics and a significant AFB1 adsorption capacity, with percentages of 55.70% and 51.23% at concentrations of 25 and 50 ng/mL, respectively, showing no statistical difference between these concentrations. In comparison, Pinheiro et al. (2020) evaluated three yeast strains at AFB1 concentrations of 10 and 25 ng/mL and reported adsorption percentages of 21.19% and 19.77%. In their study, the strain A8L2 was identified as the most effective; however, its adsorption values were considerably lower than those obtained for the A8L3 strain in the present research.

The results obtained indicate the feasibility of including this strain in Nile tilapia feed while reducing the amount of AFB1 in the feed occasionally ingested by these animals, which can lead to an improvement in physiological and sanitary aspects. Therefore, in vivo studies with these animals are necessary to obtain a better assessment of fish farming management.

4. Conclusion

The present study shows that the yeast strain Saccharomyces cerevisiae A8L3 isolated from fish farms has probiotic and aflatoxin B1 adsorption capacity in in vitro tests. With all these desirable characteristics for a probiotic, this strain meets the analyzed requirements to be used in the formulation of diets for use in in vivo trials in fish farming.

Acknowledgements

To the Center for Study, Research and Food Processing (NUEPPA), of the Agricultural Sciences Center of the Federal University of Piauí for the facilities and support in this research and to the Coordination for the Improvement of Higher Education Personnel (CAPES) for assistance with the scholarship throughout this period of doctorate.

Data Availability Statement

The datasets generated and analyzed during the current study are available from the corresponding author, E. J. R. Santos, upon reasonable request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  17 Apr 2026
• Date of issue
  2026

History

• Received
  29 Oct 2025
• Accepted
  04 Mar 2026