鸡肠道微生物代谢黄曲霉毒素B1的分子机制研究

The aflatoxin B1 is recognized as the most dangerous mycotoxins which is extremely toxic and the greatest harm. Clarify the metabolic mechanism of AFB1 in vivo is fundamental for controlling toxins. Our Group had carried out research on the metabolism of AFB1 in liver in recent years, but the metabolism of AFB1 by gut microbiota is not clear. There are a large number of microorganisms in animal intestine called as acquired the mysterious "organ", which metabolize many kinds of xenobiotics. Chicken are selected as the research object to make clear the metabolism of AFB1 by gut microbe. In our previous study, a AFB1 degradation strain CG008 was separated from intestines, and its metabolites did not show any toxicity. We plan to screen and identify the key enzymes which are responsible for the AFB1 degradation by constructing a cDNA library or protein analysis. Then analysis with mass spectrometry (MS), infra-red spectroscopy (IR), nuclear magnetic resonance (NMR) show the molecular structure of metabolites of AFB1, and determination the actual conversion rate of AFB1 metabolites in vivo. The results of this study are benefit to clarified the metabolic mechanism of AFB1 in vivo, and provide theoretical guidance and evidence for AFB1 biological detoxification also.

黄曲霉毒素B1（Aflatoxin B1, AFB1）是世界公认的毒性最强、危害最大的真菌毒素。明确AFB1在宿主体内的代谢机制，是控制毒素危害的重要前提。本课题组近年来已开展了AFB1在动物肝脏内的代谢研究，但其在肠道中的代谢情况还不清楚。动物肠道中聚集着数量庞大的微生物群，能代谢多种外源物质，被称为后天获得的神秘“器官”。本项目拟以鸡肠道微生物为研究对象，研究其对AFB1的代谢转化作用。前期实验已从鸡肠道中分离到一株高效、稳定的AFB1代谢菌CG008，并证实其代谢产物无细胞毒性。下一步拟通过构建cDNA表达文库或者通过蛋白质等分析技术获得代谢活性物质并做功能验证；采用质谱、红外光谱、核磁共振等技术分析代谢产物的分子结构；最后通过体内试验，确定鸡肠道中AFB1的实际转化情况。本项目有助于阐明AFB1在动物体内的代谢机制，同时也为AFB1的生物减毒提供理论依据。

黄曲霉毒素B1（Aflatoxin B1, AFB1）是世界公认的毒性最强、危害最大的真菌毒素，可以引起人和动物的肝脏癌变。利用微生物及其分泌的酶等降解黄曲霉毒素的生物脱毒法，具有特异性强、效率高以及对环境没有污染等优点，是未来黄曲霉毒素脱毒的主要发展方向。本项目从鸡肠道中分离到的大肠杆菌CG1061能稳定、高效地降解AFB1，降解率为93.7%。该菌株胞外上清的AFB1降解率为61.8%，胞内提取物的降解率为17.6%，说明其主要降解活性物质是分泌到细胞外的。蛋白酶处理胞外上清，降解率由61.8%下降至37.5%；100℃处理20min，降解率降低为51.3%，初步推测降解活性物质是一种耐热的蛋白酶。探索胞外上清降解AFB1的最佳作用条件，结果表明pH、温度和时间分别为8.5，55℃和24h时降解率最高。胞外上清用硫酸铵沉淀后，采用离子交换柱、葡聚糖凝胶柱等分离纯化手段，但始终没有得到纯化的降解酶。UPLC Q-TOF MS分析降解产物，结果表明AFB1可能被生物转化为AFD1 (C16H14O5)。鸡肝细胞和小鼠注射实验对比AFB1代谢前和代谢后的生物毒性，从细胞水平和动物水平上均证实代谢产物毒性大大降低。本项目成功从健康鸡肠道中分离到一株AFB1解毒菌，并对其降解特性、关键酶和代谢产物等做了深入研究，为AFB1污染饲料和食品原料的生物减毒提供了优良菌种资源和实验依据。