本实验采用富集培养的方法从土壤中筛选出有效降解玉米赤霉烯酮（Zearalenone，ZEN）的菌株。初筛采用ZEN涂布至无机盐培养基作为唯一碳源，复筛是以菌株的发酵液对ZEN的降解率作为评价指标，得到一株菌株A.lwoffi.Haut.1，其发酵液在37℃下与5 μg/mL ZEN共培养48 h，降解率高达93.54%。经16S rDNA基因测序分析、生理生化实验和菌落形态观察初步对菌株进行鉴定，并对该菌株的降解活性物质进行初步定位，最终探究丹宁-聚乙二醇法和盐沉法对无细胞上清液进行粗降解酶的提取效果。结果表明，该菌株鉴定判断为鲁氏不动杆菌（Acinetobacter Lwoffii）；该菌株的无细胞上清液的降解率最高为82.31%，无细胞上清液经加热处理、蛋白酶K处理和蛋白酶K+SDS处理后，降解率分别为20.10%、41.67%和18.68%，说明降解活性物质主要为胞外酶；当单宁浓度为15 mg/mL，聚乙二醇溶液浓度为10 mg/mL，提出的酶液降解率为64.33%，而盐沉法加入60%硫酸铵提出的粗酶液降解率为20.30%，因此，单宁聚乙二醇法提取降解酶效果更好。本研究为菌株降解ZEN的作用机理提供了研究基础，也为生物降解ZEN提供了新的研究材料。

In this experiment, Zearalenone (ZEN) -degrading strain was screened from soil by enrichment culture method. ZEN was coated to inorganic salt medium as the sole carbon source in initial screening, and the degradation rate of ZEN in the fermentation broth of the strain was used as the evaluation index in the second screening. The strain with the highest degradation rate was named A.lwoffi.Haut.1, which was identified by physiological and biochemical experiments and 16S rDNA gene sequence analysis. Then the degradation active substances of the strain were preliminarily located. ZEN-degrading enzyme in cell-free supernatant was preliminarily isolated by tannic acid - polyethylene glycol method and salt precipitation method. A.lwoffi.Haut.1 was identified as Acinetobacter Lwoffii by physiological and biochemical analysis and 16S rDNA gene sequence analysis. The degradation rate of 5 μg/mL ZEN reached 93.54% after incubating with fermentation broth for 48h at 37℃. The ZEN degradation activity of A.lwoffi.Haut.1 was mainly derived from cell-free supernatant which had a degradation rate of 82.31%. The degradation rate of cell-free supernatant was reduced to 20.10%, 41.67% and 18.68% by heating, protease K and protease K+SDS treatment, respectively. The results showed that extracellular enzymes were the main degradation active substances. After 15 mg/mL tannic acid treatment, the degradation rate of supernatant decreased to 12.33%. When the tannin-enzyme complex was dissolved in 10 mg/mL polyethylene glycol solution, the degradation rate of supernatant after centrifugation was 64.33%. Only 20.30% degradation rate was obtained from the crude enzyme solution screened by ammonium sulfate precipitation. Therefore, tannin-polyethylene glycol has a better effect on the extraction of degrading enzymes. This study provides a research basis for the bacterial degradation mechanism of ZEN and a new research materials for the biodegradation.

2020年度河南省高校科技创新团队:食品营养安全检测与控制(20IRTSTHN023); 粮油食品加工过程中真菌毒素控制关键技术研发及应用(2020CXZX0077);