淡豆豉中产γ-氨基丁酸微生物对产毒黄曲霉菌的拮抗作用和毒素合成关键基因mRNA表达的影响

目的 考察从淡豆豉Sojae Semen Praeparatum（SSP）炮制过程中分离的15株产γ-氨基丁酸微生物对产毒黄曲霉菌的拮抗能力并筛选出具有抑制产毒黄曲霉菌生长和降解黄曲霉毒素B₁（aflatoxin B₁，AFB₁）的高效拮抗菌，探究高效拮抗菌发酵产物对黄曲霉毒素（aflatoxins）合成关键基因mRNA表达量的影响。方法 运用平板对峙法与十字交叉法检测15株产γ-氨基丁酸微生物及其发酵产物对产毒黄曲霉标准株（简称：产毒标准株）生长的影响，超高效液相色谱-串联质谱法（ultra performance liquid chromatography mass spectrometry，UPLC-MS/MS）检测15株菌对AFB₁的降解效果，考察其对产毒黄曲霉菌的拮抗能力并筛选出高效拮抗菌；针对高效拮抗菌发酵产物分别运用菌丝干重法检测其对产毒标准株菌丝生长的影响，实时荧光定量PCR（real-time quantitative polymerase chain reaction，RT-qPCR）检测其对黄曲霉毒素合成关键基因（aflR、aflD、aflM、aflP）mRNA表达的影响。结果 15株菌及其发酵产物对产毒标准株的生长抑制率分别在15.88%～56.05%和25.74%～52.12%，对AFB₁降解率在2.74%～57.87%，表明它们对产毒黄曲霉菌均有一定的拮抗作用，并筛选出具有高效拮抗作用的黑曲霉菌Aspergillus niger（JC2）和枯草芽孢杆菌Bacillus subtilis（Xd1）。当高效拮抗菌发酵产物加入量为2 000 µL时，JC2和Xd1对产毒标准株菌丝生长的抑制率分别为73.82%和63.34%，且能明显抑制黄曲霉毒素合成关键基因aflR、aflD、aflM、aflP的mRNA表达。结论 淡豆豉炮制中存在既能产γ-氨基丁酸又能明显抑制产毒标准株生长和产毒的拮抗菌，其拮抗作用可能通过抑制产毒标准株菌丝生长和毒素合成关键基因的mRNA表达。

Antagonistic effects of γ-amino butyri acid-producing microorganisms existing in the fermentation process of Sojae Semen Praeparatum on toxigenic Aspergillus flavus and effects on the mRNA expression of key genes for toxin synthesis

Objective To investigate the antagonistic ability of 15 strains of γ-amino butyri acid-producing microorganisms isolated from the fermentation process of Sojae Semen Praeparatum (SSP) against toxigenic Aspergillus flavus, screened out the efficient antagonistic microorganisms with the ability to inhibit the growth of toxigenic A. flavus and degrade aflatoxin B₁ (AFB₁), and explore the effect of efficient antagonistic microorganisms fermentation products on the mRNA expression of key genes for aflatoxin synthesis. Methods The effect of 15 strains of γ-amino butyri acid-producing microorganisms and their fermentation products on the growth of toxigenic A. flavus standard strain (abbreviation: toxigenic standard strain) were tested through the plate confrontation culture and cross over method, respectively; Ultra performance liquid chromatography mass spectrometry (UPLC-MS/MS) was used to investigate the degradation effect of 15 strains on AFB₁. The antagonistic ability of 15 strains against toxigenic A. flavus was also examined and the efficient antagonistic microorganisms were screened out. For the efficient antagonistic microorganisms fermentation products, the mycelium dry weight method was used to detect its effect on the mycelium growth of the toxigenic standard strain, and real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect its effect on the mRNA expression of key genes (aflR, aflD, aflM, aflP) for aflatoxin synthesis. Results The growth of the toxigenic standard strain was inhibited by the 15 strains and their fermentation products, with inhibition rates between 15.88%—56.05% and 25.74%—52.12%, respectively. The degradation rate of AFB₁ was between 2.74%—57.87%. It was shown that they all have certain antagonistic effects on toxigenic A. flavus. In addition, Aspergillus niger (JC2) and Bacillus subtilis (Xd1) with efficient antagonistic effects were screened out. When the addition amount of highly efficient antagonistic microorganism fermentation product was 2 000 μL, the inhibition rates of JC2 and Xd1 on the mycelium growth of toxigenic standard strain were 73.82% and 63.34%, respectively, and the mRNA expression of aflatoxin synthesis key genes aflR,aflD, aflM and aflP could be significantly inhibited. Conclusion There are antagonistic microorganism in the fermentation process of SSP that can not only produce γ-amino butyri acid, but also significantly inhibit the growth and toxin production of toxigenic standard strain. Its antagonistic effect may be achieved by inhibiting the mycelium growth of toxigenic standard strain and the mRNA expression of key genes for toxin synthesis.