Detoxification of Aflatoxin B1 by a Potential Probiotic Bacillus amyloliquefaciens WF2020

Abstract

Microbial degradation is considered as an attractive method to eliminate exposure to aflatoxin B1 (AFB1), the most toxic mycotoxin that causes great economic losses and brings a serious threat to human and animal health, in food and feed. In this study, Bacillus amyloliquefaciens WF2020, isolated from naturally fermented pickles, could effectively degrade AFB1 ranging from 1 to 8 μg/ml, and the optimum temperature and pH value were 37-45°C and 8.0, respectively. Moreover, B. amyloliquefaciens WF2020 was considered to be a potential probiotic due to the synthesis of active compounds, absence of virulence genes, susceptibility to various antibiotics, and enhanced lifespan of Caenorhabditis elegans. Extracellular enzymes or proteins played a major role in AFB1 degradation mediated by B. amyloliquefaciens WF2020 into metabolites with low or no mutagenicity and toxicity to C. elegans. AFB1 degradation by the cell-free supernatant was stable up to 70°C, with an optimal pH of 8.0, and the cell-free supernatant could still degrade AFB1 by 37.16% after boiling for 20 min. Furthermore, B. amyloliquefaciens WF2020 caused a slight defect in fungal growth and completely inhibited AFB1 production when co-incubated with Aspergillus flavus. Additionally, B. amyloliquefaciens WF2020 suppressed the expression of 10 aflatoxin pathway genes and 2 transcription factors (alfR and alfS), suggesting that B. amyloliquefaciens WF2020 might inhibit AFB1 synthesis in A. flavus. These results indicate that B. amyloliquefaciens WF2020 and/or its extracellular enzymes or proteins have a promising potential to be applied in protecting food and feed from AFB1 contamination.

Keywords: Ames test; Aspergillus flavus; Bacillus amyloliquefaciens; Caenorhabditis elegans; aflatoxin B1; genome sequence.

FIGURE 1

Aflatoxin B1 (AFB1) degradation mediated by Bacillus amyloliquefaciens WF2020 at different concentrations of AFB1 (A) and circular representation of the complete genome of B. amyloliquefaciens WF2020 (B). From outermost to innermost circle: circle 1, genome size; circle 2, genes on forward strand; circle 3, genes on reverse strand; circle 4, rRNA and tRNA; circle 5, GC content; and circle 6, GC skew.

FIGURE 2

AFB1 degradation among diverse cell components of B. amyloliquefaciens WF2020. (A) AFB1 degradation by extracellular extracts, intracellular extracts, and dead cells during 72-h incubation with 2 μg/ml AFB1 at 37°C. (B) Effects of heat, proteinase K (PK), SDS, and proteinase K plus SDS on AFB1 degradation mediated by the cell-free supernatant after co-incubation for 24 h. (C–E) Effects of different temperatures (C), pH values (D), and metal ions (E) on AFB1 degradation mediated by the cell-free supernatant after co-incubation for 48 h. Different lowercase letters in the bars of each group indicate significant differences between treatments (Tukey’s test, p < 0.05).

FIGURE 3

The safety of B. amyloliquefaciens WF2020 and its AFB1 degradation products. (A) Susceptibility of B. amyloliquefaciens WF2020 to different antibiotics by the disk diffusion test. (B) Changes in the lifespan of C. elegans N2 caused by B. amyloliquefaciens WF2020. (C,D) Reduction of AFB1 mutagenic effects (C) and the toxicity to C. elegans N2 (D) caused by B. amyloliquefaciens WF2020. The AFB1 group means extracts from the media supplemented with 20 μg AFB1. The DM group refers to the culture extracts from the supernatant of the 96 h co-incubation of 20 μg AFB1 and B. amyloliquefaciens WF2020. The CN group means the control group. Different lowercase letters in the bars of each group indicate significant differences between treatments (Tukey’s test, p < 0.05).

FIGURE 4

Effects of temperature (A,B), initial pH value (C,D), and metal ions (E,F) on the AFB1 degradation (A,C,E) and bacterial growth (B,D,F) in B. amyloliquefaciens WF2020. In terms of temperature, the residual AFB1 was analyzed after B. amyloliquefaciens WF2020 was co-incubated with 2 μg/ml AFB1 for 72 h. With respect to the effects of initial pH value and metal ions, the residual AFB1 was analyzed after B. amyloliquefaciens WF2020 was co-incubated with 2 μg/ml AFB1 at 37°C for 24 and 48 h, respectively. Different lowercase letters on the bars of each group indicate significant differences between the treatments (Tukey’s test, p < 0.05).

FIGURE 5

Changes in fungal growth, AFB1 production, and transcriptional expression of genes involved in AFB1 synthesis in Aspergillus flavus. (A) The antagonistic effect of B. amyloliquefaciens WF2020 on fungal growth after the bacterium was co-incubated with A. flavus for 2 days on a plate of potato dextrose agar (PDA). (B,C) The reduction of dry weight (B) and AFB1 production (C) caused by B. amyloliquefaciens WF2020 after the bacterium was co-incubated with A. flavus for 2 days in the potato dextrose broth (PDB). (D) Quantitation of relative transcriptional levels of selected genes associated with AFB1 synthesis in A. flavus via real-time quantitative polymerase chain reaction (qRT-PCR) after B. amyloliquefaciens WF2020 was co-incubated with A. flavus for 2 days in PDB. The line represents the transcriptional levels of genes in control experiments, which were defined as 1. Different lowercase letters in the bars of each group indicate significant differences between treatments (Tukey’s test, p < 0.05).