1株降解黄曲霉毒素B1的解淀粉芽胞杆菌的分离鉴定及其体外脱毒效果

旨在筛选出能够高效降解黄曲霉毒素B₁（AFB₁）的菌株，并对其脱毒活性组分的脱毒效果进行研究，本研究以香豆素为唯一碳源对目标细菌进行初筛，以对AFB₁的降解率为指标进行复筛，并从形态学、生理生化试验和16S rDNA序列分析对筛选菌株进行鉴定；通过测定该菌株不同发酵组分对AFB₁的降解率，来定位其脱毒活性组分，同时采用CCK-8法测定活性组分降解AFB₁后对LMH细胞毒性，并研究了热处理、紫外线照射、强酸、强碱及蛋白酶K处理对活性组分脱毒作用的影响。结果显示，从样品中初筛分离到24株能够在初筛培养基生长良好的菌株，经复筛从中筛选到一株能高效降解AFB₁的菌株Y1-B1，其AFB₁降解率达到73.2%；经形态学、生理生化试验和16S rDNA序列分析确定该菌为解淀粉芽胞杆菌（Bacillus amyloliquefaciens）。对菌株Y1-B1培养物的不同组分进行的脱毒活性研究表明，不同组分对AFB₁的降解率不同，上清液的脱毒能力最强，细菌细胞裂解物的脱毒能力下降明显，菌体悬液和菌体裂解物上清的脱毒能力最差，由此确定解淀粉芽胞杆菌Y1-B1的脱毒能力主要来源于上清液中的某种活性物质；细胞毒性结果显示，与AFB₁对LMH细胞的毒性作用相比，AFB₁被上清液降解后其产物的毒性显著降低。该脱毒活性组分对不同理化因素处理呈现出不同的表现，对高温、紫外照射抵抗力较强，对强酸、强碱抵抗力较差，蛋白酶K仅造成脱毒能力部分减弱。本研究将为解决黄曲霉毒素污染问题提供新的微生物种质资源，同时为后续微生物脱毒制剂的开发奠定基础。

Isolation and Identification of Aflatoxin B1 Degrading Bacillus amyloliquefaciens and Its in vitro Detoxification Effect

The aim of this study is to screen out strains that can efficiently degrade aflatoxin B₁ (AFB₁), and to investigate the detoxification effect of its detoxification active components. In this study, the target bacteria were primarily screened by coumarin as the sole carbon source, and rescreened based on the degradation ratio of AFB₁.The selected strain was identified by morphological observation, physiological and biochemical tests, and 16S rDNA sequence analysis. The detoxification active components produced by the bacteria were located by measuring the AFB₁ degradation rate of the different fermentation components. The cytotoxicity of AFB₁degradation products to LMH cells was analyzed by CCK-8 method. And the effects of heat treatment, ultraviolet radiation, strong acid, strong alkali and proteinase K treatment on the detoxification effects of active ingredients were also studied. The results showed that 24 bacteria strains could grow well in the primary screening medium. After detoxification re-screening, strain Y1-B1 could efficiently degrade AFB₁, and its degradation ratio of AFB₁ reached 73.2%. Through morphological observation, physiological and biochemical tests and 16S rDNA sequence analysis, this strain Y1-B1 was identified as Bacillus amyloliquefaciens. Studies on the detoxification activity of different fermentation components of the cultures of strain Y1-B1 showed different degradation rate of AFB₁, with the supernatant of cell culture exhibited the strongest detoxification activity, followed by bacterial lysates, suspension of bacteria, and supernatant of bacterial lysate. The results demonstrated that the detoxification active components of Bacillus amyloliquefaciens Y1-B1 was an extracellular product in the supernatant. The results of cytotoxicity showed that the toxi-city of AFB₁ degraded by the detoxification active components was significantly reduced compared with the toxicity of AFB₁ to LMH cells. The detoxification active components present different performances to different physical and chemical factors, and has strong resistance to high temperature and ultraviolet radiation, relatively poor resistance to strong acids and strong bases, and proteinase K only partially weakens the detoxification ability. This study will provide new microbial germplasm resources for solving the problem of aflatoxin pollution and also lay foundation for the development of subsequent microbial detoxification products.