枯草芽孢杆菌ANSB01G对饲料中玉米赤霉烯酮生物降解机制的研究

Zearalenone (ZEN) is a non-steroidal estrogen produced by many Fusarium species in cereals and other plants,and is frequently implicated in safety of foods and feeds. ZEN can irreversibly combine with the estrogens receptor in uterus so that reproduction physiology of animal are influenced,which lead to enormous hazards on livestock breeding. Several physical and chemical strategies for the elimination or inactivation of ZEN have been reported in the literature. Nevertheless, none of these strategies completely fulfills the necessary efficacy, safety, and cost requirements, and none is entirely effective. In recent years, biological detoxification of ZEN by microbes and biological enzyme have been extensively investigated because of its advantages contained high detoxification efficiency,strong specificicy and transferring mycotoxin into notoxic metabolites in moderate condition and so on. We have isolated a strain of Bacillus subtilis from intestinal tract chymus of chicks, which can degraded over 75% ZEN. At the same time, we have determined the effect(s) of crude protein extracts from supernate fluid of Bacillus subtilis on ZEN is enzymatic in nature. An invention patent on this strain has been applied. The patent number is authorized as ZL 201010620651.2. This research will isolate and purify the pure ZEN degrading enzyme from supernate fluid of Bacillus subtilis and investigate the main characteristics of this enzyme. Biodetoxification mechanism on ZEN in feed by this enzyme will be studied with HPLC and LC-MS Q-TOF. Effects of degrading ZEN will be studied with artificial simulated duodenal and gastric juice, which will lay the theoretical foundation for exploiting the efficient and safe feed additive of mycotoxin degradation enzyme.

玉米赤霉烯酮(ZEN)具有类雌激素作用，可与子宫内雌激素受体不可逆结合而影响动物的生殖生理，对养殖业造成巨大危害。传统的物理化学去毒方法因其存在效果不稳定、营养成分损失大、影响饲料适口性等缺点而不能被广泛应用到实际生产中。微生物及生物酶解毒因具有解毒效率高、特异性强、可在温和条件下将毒素转化为无毒代谢产物等优点备受研究者关注。本课题组已从鸡肠道食糜中分离筛选到一株可高效降解ZEN的枯草芽孢杆菌,初步研究确定起解毒作用的活性物质是一种细菌所产胞外酶。该菌已被申请国家发明专利并获授权（专利号：ZL201010620651.2）。本项目将对枯草芽孢杆菌发酵产物中的降解酶进行分离纯化及酶学特性进行研究；用HPLC和LC-MS Q-TOF研究其对饲料中ZEN生物降解作用机理；人工模拟肠胃液体外验证酶降解ZEN的作用效果，为研发高效、安全霉菌毒素降解酶饲料添加剂奠定理论基础。

玉米赤霉烯酮（ZEA）具有类雌激素作用，可与子宫内雌激素受体不可逆结合而影响动物的生殖生理，对养殖业造成巨大危害。传统的物理化学去毒方法因其存在效果不稳定、营养成分损失大、影响饲料适口性等缺点而不能被广泛应用到实际生产中。微生物及生物酶解毒因具有解毒效率高、特异性强、可在温和条件下将毒素转化为无毒代谢产物等优点备受研究者关注。本课题组已从鸡肠道食糜中分离筛选到一株可高效降解ZEA的枯草芽孢杆菌ANSB01G，其发酵上清液对ZEA的降解率大于75%，初步研究确定起解毒作用的活性物质是一种胞外酶。本项目拟对枯草芽孢杆菌发酵产物中的降解酶进行分离纯化及酶学特性研究；用HPLC和LC-MS Q-TOF研究其降解ZEA的作用机理；人工模拟肠胃液体外验证酶降解ZEA的作用效果。.取得的主要成果：菌株ANSB01G发酵产生的ZEA降解酶粗蛋白经硫酸铵沉淀，离子交换层析、疏水层析和分子筛层析后，再进行SDS-PAGE 和MALDI-TOF/TOF/MS分析，最终得到分子量为55kDa的Catalase蛋白。酶学特性研究表明，降解酶在温度为30～40˚C，pH为7.5～8.5之间有较高的酶活，其中最佳酶活反应条件是温度35˚C，pH为8.5；Km为1.29×10-5 mol/L，具有良好的酶动力学特性。通过高效液相色谱-二级质谱联用测定ZEA降解产物的分子结构，推测菌株降解ZEA的作用机制是在酶的作用下，ZEA酚羟基与谷氨酸的γ-羧基结合，形成初级代谢产物M1；然后ZEA的内酯环水解，脱羧，并还原羰基、脱水，产生次级代谢产物M2。通过建立体外模拟消化系统，测定了降解酶对ZEA的降解率，结果显示在体外模拟消化系统中降解酶对饲料中ZEA的降解率为66.80%。试验结果为研发高效、安全霉菌毒素降解酶饲料添加剂奠定理论基础。发表SCI论文4篇，中文核心期刊文章1篇，获授权国家发明专利1项，中华农业科技奖1项。.