丁酸梭菌发酵培养基的优化及发酵产物对黄曲霉毒素B1的降解

Fermentation medium optimization of Clostridium butyricum and degradation of aflatoxin B1 by fermentation products

摘要:

【背景】丁酸梭菌是专性厌氧的新一代芽孢益生菌，耐热、耐酸、抗逆性强，极具应用价值和开发前景。【目的】优化丁酸梭菌发酵培养基并初步研究其发酵液对黄曲霉菌的抑制作用和降解黄曲霉毒素B₁ (aflatoxin B₁, AFB₁)的能力。【方法】利用响应面法对发酵培养基进行优化，采用牛津杯法对丁酸梭菌发酵液抑制黄曲霉菌生长进行研究，并通过酶联免疫法测定发酵液对AFB₁的降解能力。【结果】优化后的发酵培养基为：葡萄糖18.1 g/L，大豆蛋白胨29.7 g/L，磷酸氢二钾3.8 g/L，氯化钠2.0 g/L，乙酸钠4.0 g/L，结晶硫酸镁1.2 g/L，l-半胱氨酸盐酸盐0.3 g/L。优化后的丁酸梭菌生物量由8.99×10⁸个/mL提高至2.28×10⁹个/mL，是优化前的2.54倍。丁酸梭菌发酵液对致病真菌黄曲霉菌的抑菌效果十分显著，其上清液经浓缩后对AFB₁降解72 h的降解率达到68.65%，初步分析表明上清液中对AFB₁具有脱毒作用的活性组分为丁酸梭菌分泌产生的胞外酶。【结论】本研究通过发酵培养基优化明显提高了丁酸梭菌的生物量，并将其应用于抑制黄曲霉菌的生长与降解AFB₁，为丁酸梭菌的规模化生产及其微生态制剂的开发应用提供了科学依据。

Abstract:

[Background] Clostridium butyricum is a new generation of obligate anaerobic spore-producing probiotics with strong tolerance to heat, acid, and stress, demonstrating a great application value and development prospect. [Objective] To optimize the fermentation medium of C. butyricum and study the activities of the fermentation broth in terms of inhibiting Aspergillus flavus and degrading aflatoxin B₁ (AFB₁). [Methods] Response surface methodology was employed to optimize the fermentation medium. The oxford cup assay was employed to examine the inhibitory activity of C. butyricum fermentation broth on A. flavus and enzyme-linked immunosorbent assay to determine the AFB₁-degrading ability of the fermentation broth. [Results] The optimized fermentation medium was composed of 18.1 g/L glucose, 29.7 g/L soya peptone, 3.8 g/L K₂HPO₄· 3H₂O, 2.0 g/L NaCl, 4.0 g/L NaAc, 1.2 g/L MgSO₄· 7H₂O, and 0.3 g/L l-cystine hydrochloride. After optimization, the biomass of C. butyricum reached 2.28×10⁹ cells/mL, which was 2.54 times of that (8.99×10⁸ cells/mL) before optimization. The fermentation broth of C. butyricum exerted a significant inhibitory effect on A. flavus, and the concentrated supernatant degraded 68.65% of AFB₁ within 72 h. The results indicated that the active components degrading AFB₁ in the supernatant were the extracellular enzymes produced by C. butyricum.[Conclusion] The biomass of C. butyricum was significantly increased by fermentation medium optimization, and the fermentation broth of C. butyricum inhibited A. flavus and degraded AFB₁. The findings provide scientific evidence for the industrial production of C. butyricum and the development and application of microecological preparations.