Investigating the mechanism of Bacillus amyloliquefaciens YUAD7 degrading aflatoxin B1 in alfalfa silage using isotope tracing and nuclear magnetic resonance methods

Background Fungal toxins are highly toxic and widely distributed, presenting a considerable threat to global agricultural development. Addressing the issue of aflatoxin B-1 (AFB(1)) contamination in feed, it is crucial to ascertain the effectiveness and mechanisms of microbial strains in degradation. Results This study used isotope tracing and nuclear magnetic resonance (NMR) to investigate the degradation products of Bacillus amyloliquefaciens YUAD7 in complex substrates. By tracing 14C34-AFB1 and utilizing NMR, ultra-performance liquid chromatography-quadrupole time-of-flight/mass spectrometry (UPLC-Q-TOF/MS) purification and identification techniques, it was confirmed that AFB1 was degraded by YUAD7 into C12H14O4, C5H12N2O2, C10H14O2, and C4H12N2O, effectively removing 99.7% of AFB1 (100 mu g/kg) from alfalfa silage. YUAD7 targeted the ester bond in the vanillin lactone ring structure, the ether bond in the furan ring structure, and the unsaturated carbon-carbon double bond in the furan ring structure during AFB1 degradation, disrupting the toxic sites responsible for AFB1's carcinogenic, teratogenic, and mutagenic effects and achieving biodegradation. Moreover, B. amyloliquefaciens YUAD7 transformed AFB1 through processes like hydrogenation, enzyme modification, and the loss of the -CO group while also being associated with metabolic pathways such as alanine, aspartate, glutamate metabolism, glutathione metabolism, cysteine and methionine metabolism, and pentose and glucuronate interconversions. Conclusions The utilization of isotope tracing allowed for rapid identification of degradation products in complex substrates, while NMR elucidated the structures of these products. This deepens our understanding of AFB1 biodegradation mechanisms, providing technical support for the practical application of these bacteria in degradation, and new insights into studying the biological degradation mechanism. B. amyloliquefaciens YUAD7 can be used as a potential strain for degrading AFB1 in large-scale silage.