同时降解多种真菌毒素的菌株筛选、鉴定及降解特性研究

    Screening, identification, and degradation characteristics study of bacteria capable of simultaneously degrading multiple mycotoxins

    • 摘要: 真菌毒素是丝状真菌在生长繁殖过程中产生的一类具有强烈毒性的代谢产物,严重威胁粮食安全及人类和动物健康。以玉米赤霉烯酮(zearalenone, ZEN)、黄曲霉毒素B1(aflatoxin B1,AFB1)和呕吐毒素(deoxyniralenol, DON)的混合物作为唯一碳源,旨在筛选出能够同时降解这3种毒素的微生物菌株,并进行菌种鉴定。此外,通过对菌株的降解活性物质进行定位,利用超高效液相色谱串联质谱鉴定降解产物,并优化反应条件以提高粗酶液的降解效率。结果表明:从糖蒜中获得的枯草芽孢杆菌(HNGD-sg5)能有效降解ZEN、AFB1和DON,其单一毒素降解率分别为97.27%、93.81%和49.74%,在混合毒素中处理72 h时对3种毒素的降解率分别为99.25%、98.94%和29.86%;发现HNGD-sg5通过胞外酶将3种毒素分别转化为15-OH-ZEN、AFB1-diol和3-epi-DON进行脱毒,添加Mn2+能显著增强其对ZEN和AFB1的降解活性,在50℃和pH 7的条件下粗酶液的降解活性最高。本研究证实了HNGD-sg5菌株在治理多种真菌毒素污染的饲料方面具有良好的应用潜力,为真菌毒素降解菌制剂的开发和相关降解酶基因的后续挖掘奠定了基础。

       

      Abstract: Mycotoxins are a class of highly toxic metabolites produced by filamentous fungi during their growth and reproduction, which pose serious threats to food security as well as human and animal health. In this study, a mixture of zearalenone(ZEN), aflatoxin B1(AFB1), and deoxynivalenol(DON) was utilized as the sole carbon source to screen for bacteria capable of simultaneously degrading these three toxins. Bacterial strains were identified through morphological analysis, as well as 16S rDNA and gyrB sequence analysis. By examining the degradation capabilities of bacterial fermentation broth, supernatant, cell suspension, intracellular extract, and inactivated bacterial cells, we aimed to explore the degradation potential of bacterial strains towards three types of toxins and identify the active sites for their degradation. To investigate the degradation properties of the active substances, proteinase K, SDS, and heat treatments were applied to the supernatant. Degradation products were identified using Ultra-High Performance Liquid Chromatography coupled with Tandem Mass Spectrometry(UHPLC-MS/MS). Additionally, optimization of the reaction conditions was conducted by exploring the effects of various metal ions, fermentation temperatures, and pH values to enhance the degradation efficiency of the enzymes. The results indicated that a strain of Bacillus subtilis HNGD-sg5, isolated from sweetened garlic, was capable of degrading ZEN, AFB1, and DON, with degradation rates of 97.27%, 93.81%, and 49.74%, respectively, and degradation rates of 99.25%, 98.94%, and 29.86% for the mixed toxins. The primary active sites for the degradation of three toxins were located in the fermentation supernatant, where the bacteria exhibited some physical adsorption for ZEN degradation. Further inactivation treatments of the supernatant revealed that the bacteria primarily degraded ZEN, AFB1, and DON into 15-OH-ZEN, AFB1-diol, and 3-epi-DON, respectively, through extracellular enzymes. These extracellular enzymes demonstrated high-temperature resistance in the degradation of AFB1. In the optimization experiment, Fe3+, Ni2+, Mn2+, and Mg2+ enhanced the degradation activity of the crude enzyme solution towards ZEN, while Mn2+, Cu2+, and Zn2+enhanced the degradation activity of the crude enzyme solution towards AFB1, only Cu2+ enhanced the degradation of DON. The degradation activity of the crude enzyme solution was highest under fermentation conditions of 50 ℃ and pH 7. These findings indicate that Bacillus subtilis HNGD-sg5 has significant potential for the simultaneous degradation of ZEN, AFB1, and DON, providing a foundation for the development of fungal toxin-degrading enzyme preparations and the subsequent exploration of related degrading enzyme genes.

       

    /

    返回文章
    返回