| 降解AFB1的枯草芽孢杆菌S1分离与鉴定 |
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| 引用本文： | 孙向丽,刘宇翾,时子瑶,温长印,田亚东,康相涛,王彦彬.降解AFB1的枯草芽孢杆菌S1分离与鉴定[J].中国畜牧兽医,2022,49(12):4654-4664. |
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| 作者姓名： | 孙向丽 刘宇翾 时子瑶 温长印 田亚东 康相涛 王彦彬 |
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| 作者单位： | 1. 河南农业大学动物科技学院, 郑州 450046;2. 河南农业大学动物医学院, 郑州 450046;3. 河南金尔康生物科技有限公司, 新乡 453000 |
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| 基金项目： | 中原科技创新领军人才项目（214200510003）；河南省高校科技创新团队支持计划（21IRTSTHN022） |
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| 摘 要： | 【目的】筛选出能够高效降解黄曲霉毒素（aflatoxin，AF）的菌株，并对其降解活性及残留毒性进行检测，以期为黄曲霉毒素B1（alflatoxin B1，AFB1）污染防治提供参考。【方法】采集耗牛、绵羊等草食性动物粪便和养殖场霉变饲料，以AFB1的结构类似物香豆素为唯一碳源筛选降解AFB1效率最高的菌株，并对其进行形态学、生理生化和16S rDNA鉴定。取AFB1与适量筛选菌株的发酵液混合，使AFB1的浓度为1 μg/mL，37℃、150 r/min培养，测定培养不同时间AFB1降解率；应用差速离心法分别制取上清液、菌悬液和菌体，将菌体超声裂解后制得胞内液，测定不同组分对AFB1的降解率；对上清液分别进行蛋白酶K、蛋白酶K+SDS和热处理，检测处理后上清液AFB1降解率；分别用不同浓度的饱和硫酸铵沉淀处理上清液，检测AFB1降解率；应用7 ku透析袋对上清液进行浓缩，检测AFB1降解率，从而判断菌株活性成分的性质和分布。将鸡肝细胞分为AFB1组、AFB1-菌株组、菌株组和空白对照组，用实时荧光定量PCR检测各组鸡肝细胞中白细胞介素-6（interleukin-6，IL-6）、肿瘤坏死因子α（tumor necrosisi factor alpha，TNF-α）和Bcl-2-associated X蛋白（Bax）基因的相对表达量。【结果】经初筛和复筛，从编号为S1的绵羊粪便样品中分离出1株菌株，其降解AFB1的活性最高，降解率可达60.36%。菌株S1在LB固体培养基上培养12 h，可观察到淡黄色不透明菌落，表面光滑，有隆起，镜检革兰氏染色呈阳性，杆状。16S rDNA基因进行PCR扩增并进一步测序分析，其序列与LC55966.1的同源性为100%。综合16 S rDNA序列分析和生理生化结果鉴定该菌为枯草芽孢杆菌（Bacillus subtilis）。该菌发酵液与AFB1（1 μg/mL）反应4、12、24、48和72 h，降解率分别达到10.98%、25.36%、46.24%和52.65%和80.84%。上清液AFB1降解率显著高于菌悬液和胞内液（P<0.05）。热处理后，上清液的AFB1降解活性降低，而经蛋白酶K和SDS处理后降解活性基本丧失。用不同浓度饱和硫酸铵处理的上清液AFB1降解率差异不显著（P>0.05），而经透析浓缩后的上清液AFB1降解率显著高于硫酸铵处理的上清液沉淀蛋白（P<0.05）。在鸡胚胎肝脏原代细胞上，与AFB1组相比，AFB1经枯草芽孢杆菌S1降解后诱导IL-6、TNF-α以及Bax的表达量均显著降低（P<0.05）。【结论】筛选到1株枯草芽孢杆菌S1，其具有较高的降解AFB1的活性，其降解活性主要来自于培养液上清中的蛋白质；AFB1经降解后其毒素显著降低。
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| 关 键 词： | 黄曲霉毒素B1 枯草芽孢杆菌 毒素降解 |
| 收稿时间： | 2022-04-26 |
Isolation and Identification of a Bacillus subtilis S1 Involved in Aflatoxin B1 Detoxification |
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| SUN Xiangli,LIU Yuxuan,SHI Ziyao,WEN Changyin,TIAN Yadong,KANG Xiangtao,WANG Yanbin.Isolation and Identification of a Bacillus subtilis S1 Involved in Aflatoxin B1 Detoxification[J].China Animal Husbandry & Veterinary Medicine,2022,49(12):4654-4664. |
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| Authors: | SUN Xiangli LIU Yuxuan SHI Ziyao WEN Changyin TIAN Yadong KANG Xiangtao WANG Yanbin |
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| Institution: | 1. College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China;2. College of Animal Medicine, Henan Agricultural University, Zhengzhou 450046, China;3. Henan Jinerkang Biotechnology Co., Ltd., Xinxiang 453000, China |
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| Abstract: | 【Objective】 This study was aimed to screen out strains that could efficiently degrade alflatoxin, and to detect the degrading activity and residual toxicity, in order to provide solutions for the prevention and control of alflatoxin B1(AFB1) pollution.【Method】 Samples were collected from feces of herbivorous animal such as yak and sheep, as well as moldy feed from livestock farms.The strain with the highest efficiency in degrading AFB1 was screened using coumarin, a structural analog of AFB1, as the sole carbon source, and the strains were identified by morphology, physiology and biochemistry and 16S rDNA sequencing.AFB1 was mixed with an appropriate amount of fermentation broth, the final concentration of AFB1 was 1 μg/mL, and AFB1 was cultured at 37 ℃ at 150 r/min, and the degradation rate of AFB1 at different time was measured.The supernatant, bacterial suspension and bacterial cells were prepared by differential centrifugation, and the bacterial cells were ultrasonically lysed to obtain an intracellular solution, and the degradation rates of different components to AFB1 were determined.The supernatant was subjected to proteinase K, proteinase K+SDS and heat treatment to detect the degradation rate of AFB1.Precipitation of the supernatant with different concentrations of saturated ammonium sulfate, and the supernatant concentrate solutinon was prepared using 7 ku dialysis bags, that were for the measurement of degradation efficiency of AFB1.So as to determine the nature and distribution of the active ingredients of the strain.Chicken hepatocytes were divided into AFB1, AFB1-strain, strain and blank control groups.The relative expression of the inflammatory factor interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α) and Bcl-2-associated X protein (Bax) genes were evaluated.【Result】 After initial screening and rescreening, one strain was isolated from the sheep feces sample numbered S1, which had the highest activity of degrading AFB1 with a degradation rate of 60.36%.The strain S1 was cultured on the LB solid medium for 12 h, and pale yellow opaque colonies could be observed, which had smooth surface and bumps, the microscopic examination was Gram-positive and rod-shaped.The 16S rDNA gene was amplified by PCR and further sequenced, and the sequence identity with LC55966.1 was 100%.Based on nucleotide sequence analysis and physiological and biochemical results, the strain was comprehensively identified as Bacillus subtilis.The bacterial fermentation broth was reacted with AFB1 (1 μg/mL) for 4, 12, 24, 48 and 72 h, and the degradation rates reached 10.98%, 25.36%, 46.24%, 52.65% and 80.84%, respectively.The degradation rate of AFB1 in the supernatant was significantly higher than that in the bacterial suspension and intracellular solution (P<0.05).The degradation activity of the supernatant was decreased after heat treatment, while the degradation activity was basically lost after treatment with proteinase K and SDS.There was no significant difference in the degradation rate of supernatants treated with different concentrations of saturated ammonium sulfate (P>0.05).However, the degradation rate of AFB1 in the supernatant after dialysis concentration was significantly higher than that in the supernatant precipitated by ammonium sulfate (P<0.05).In chicken embryonic liver primary cells, the expressions of IL-6, TNF-α and Bax induced by degradation product of AFB1 with Bacillus subtilis S1 were significantly lower than that in AFB1 group (P<0.05).【Conclusion】 The strain of Bacillus subtilis S1 was screened out, which could degrade AFB1 efficiently.Its degradation activity was from a protein mainly distributed in the culture medium supernatant, and its residual toxicity of AFB1 was significantly reduced after degradation. |
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| Keywords: | aflatoxin B1 Bacillus subtilis detoxification |
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