Screening and identification of an aflatoxin B1-degrading strain from the Qinghai-Tibet Plateau and biodegradation products analysis

Abstract

This research aimed to address the issue of aflatoxin B₁ (AFB₁) contamination, which posed severe health and economic consequences. This study involved exploring unique species resources in the Qinghai-Tibet Plateau, screening strains capable of degrading AFB₁. UPLC-Q-Orbitrap HRMS and NMR were employed to examine the degradation process and identify the structure of the degradation products. Results showed that Bacillus amyloliquefaciens YUAD7, isolated from yak dung in the Qinghai-Tibet Plateau, removed 91.7% of AFB₁ from TSB-AFB₁ medium with an AFB₁ concentration of 10 μg/mL (72 h, 37°C, pH 6.8) and over 85% of AFB₁ from real food samples at 10 μg/g (72 h, 37°C), exhibiting strong AFB₁ degradation activity. Bacillus amyloliquefaciens YUAD7's extracellular secretions played a major role in AFB₁ degradation mediated and could still degrade AFB₁ by 43.16% after boiling for 20 min. Moreover, B. amyloliquefaciens YUAD7 demonstrated the capability to decompose AFB₁ through processes such as hydrogenation, enzyme modification, and the elimination of the -CO group, resulting in the formation of smaller non-toxic molecules. Identified products include C₁₂H₁₄O₄, C₅H₁₂N₂O₂, C₁₀H₁₄O₂, C₄H₁₂N₂O, with a structure consisting of dimethoxyphenyl and enoic acid, dimethyl-amino and ethyl carbamate, polyunsaturated fatty acid, and aminomethyl. The results indicated that B. amyloliquefaciens YUAD7 could be a potentially valuable strain for industrial-scale biodegradation of AFB₁ and providing technical support and new perspectives for research on biodegradation products.

Keywords: Bacillus amyloliquefaciens YUAD7; aflatoxin B1; biological detoxification; degradation products; detoxification mechanism.

Figure 1

The strain YUAD7 degrades AFB₁ in different substrates. (A) The degradation of different concentrations of AFB₁ by strain YUAD7 in TSB-AFB₁ medium. (B) YUAD7 degrades AFB₁ in real food samples.

Figure 2

Genome characterization of the YUAD7 strain. (A) Circular representation of the Bacillus amyloliquefaciens YUAD7 complete genome. From the outmost: 1, Genome structure positive and negative strands; 2, GC Skew; 3, GC content. (B) GO analysis of annotated coding genes in B. amyloliquefaciens YUAD7 genome. From the outmost: 1, GO ID. 2, Background genes. 3, Upregulated and downregulated genes. 4, Enrichment coefficients. (C) KEGG pathway classification of annotated coding genes in B. amyloliquefaciens YUAD7 genome.

Figure 3

Identification of active substances and factors influencing the activity of strain YUAD7 in the degradation of AFB₁. (A) AFB₁ degradation by diverse cell components of Bacillus amyloliquefaciens YUAD7 during 72-h incubation with 10 μg/mL AFB₁ at 37°C. (B) Effects of heat, PK, SDS, and PK + SDS on AFB₁ degradation mediated by the cell-free supernatant after co-incubation for 72-h with 10 μg/mL AFB₁ at 37°C.

Figure 4

Toxicological results of YUAD7 degradation products. (A) Changes in the lifespan of L-02 cells caused by different treatment groups. (B) Changes in cell morphology in different treatment groups within 72 h. (C) The Ames mutagenicity assay. NC group: medium without AFB₁. EG group: medium with AFB₁ degradation liquid of YUAD7. CC group: medium with AFB₁.

Figure 5

The mass spectra and chemical formula of AFB₁ standards and the compounds in degradation solution. (A) C₁₇H₁₂O₆. (B) C₁₂H₁₄O₄. (C) C₅H₁₂N₂O₂. (D) C₁₀H₁₄O₂. (E) C₄H₁₂N₂O.

Figure 6

Structure of the main products of AFB₁ degradation by strain YUAD7. (A) C₁₂H₁₄O₄. (B) C₅H₁₂N₂O₂. (C) C₁₀H₁₄O₂. (D) C₄H₁₂N₂O.

Figure 7

The hypothetical pathway of AFB₁ degradation by YUAD7.