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 B_(1),AFB_(1))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AFB_(1)的结构类似物香豆素为唯一碳源筛选降解AFB_(1)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AFB_(1)与适量筛选菌株的发酵液混合,使AFB_(1)的浓度为1μg/mL,37℃、150 r/min培养,测定培养不同时间AFB_(1)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AFB_(1)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AFB_(1)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AFB_(1)降解率;应用7 ku透析袋对上清液进行浓缩,检测AFB_(1)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AFB_(1)组、AFB_(1)-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AFB_(1)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AFB_(1)(1μg/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AFB_(1)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AFB_(1)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AFB_(1)降解率差异不显著(P>0.05),而经透析浓缩后的上清液AFB_(1)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AFB_(1)组相比,AFB_(1)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AFB_(1)的活性,其降解活性主要来自于培养液上清中的蛋白质;AFB_(1)经降解后其毒素显著降低。
【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 B_(1)(AFB_(1)) 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 AFB_(1) 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 16 S rDNA sequencing.AFB1was mixed with an appropriate amount of fermentation broth, the final concentration of AFB1was 1 μg/mL,and AFB_(1) was cultured at 37 ℃ at 150 r/min, and the degradation rate of AFB1at 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 AFB1were determined.The supernatant was subjected to proteinase K,proteinase K+SDS and heat treatment to detect the degradation rate of AFB_(1).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 AFB_(1).So as to determine the nature and distribution of the active ingredients of the strain.Chicken hepatocytes were divided into AFB1,AFB_(1)-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 AFB1with a degradation rate of 60.36%.The strain S1was 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 16 S 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 AFB_(1)(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 AFB_(1) 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 AFB_(1) 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 AFB_(1) with Bacillus subtilis S1were significantly lower than that in AFB1group(P<0.05).【Conclusion】 The strain of Bacillus subtilis S1was screened out, which could degrade AFB1efficiently.Its degradation activity was from a protein mainly distributed in the culture medium supernatant, and its residual toxicity of AFB1was significantly reduced after degradation.
作者
孙向丽
刘宇翾
时子瑶
温长印
田亚东
康相涛
王彦彬
SUN Xiangli;LIU Yuxuan;SHI Ziyao;WEN Changyin;TIAN Yadong;KANG Xiangtao;WANG Yanbin(College of Animal Science and Technology,Henan Agricultural University,Zhengzhou 450046,China;College of Animal Medicine,Henan Agricultural University,Zhengzhou 450046,China;Henan Jinerkang Biotechnology Co.,Ltd.,Xinxiang 453000,China)
出处
《中国畜牧兽医》
CAS
北大核心
2022年第12期1554-1564,共11页
China Animal Husbandry & Veterinary Medicine
基金
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)。
