Culture-dependent and culture-independent approaches to reveal the aflatoxin B1-producing fungi in Pixian Doubanjiang, a typical condiment in Chinese cuisine

Abstract

In Doubanjiang (DBJ) manufacturing, fungi play a critical role in shaping the unique flavor but with the risk related to mycotoxin contamination. The present study aimed to examine the occurrence of major mycotoxins and to evaluate mycotoxigenic fungi, especially aflatoxin B₁ (AFB₁)- producing fungi in DBJ products. A total of 150 samples were detected to report the occurrence of four aflatoxins (AFs) and ochratoxin A (OTA) by high performance liquid chromatography (HPLC) coupled with a fluorescence detector. The potential mycotoxin-producing fungi were isolated from AFB₁ positive samples and underwent morphological and molecular identifications. Then, the capabilities of Aspergillus flavus isolates in producing AFB₁ were evaluated using real-time PCR and HPLC. Meanwhile, the fungal communities of AFB₁ positive and negative samples were compared by next generation sequencing (NGS). In our study, the positive rates for AFB₁, AFG₁, AFB₂, AFG₂, and OTA among DBJ samples were at: 34.0%, 21.3%, 36.7%, 11.3% and 11.3%, respectively. A total of 20 A. flavus were isolated from DBJ samples, and 65% (n = 13) were verified to produce AFB₁. The NGS results have shown that the abundance of Aspergillus and A. flavus was significantly higher in AFB₁ positive samples. The findings in our study jointly suggest that A. flavus is the core AFB₁-producing fungus in DBJ manufacturing.