黄曲霉毒素B_1降解菌的筛选鉴定
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摘要
黄曲霉毒素的化学结构与香豆素十分类似,具有极强的毒性、致癌性,被认为是饲料和食品中不可避免的污染物。对黄曲霉毒素污染的控制亟须一种安全、高效、环保的脱毒方法。以香豆素作为唯一碳源的培养基筛选黄曲霉毒素B_1降解菌,最终筛选出1株对AFB_1降解率可达到91.86%的菌株(HH-1),对其进行了16S rDNA基因序列测定,系统发育进化法显示其为地衣芽孢杆菌。将地衣芽孢杆菌发酵液分为上清液和菌悬液,验证各组分对黄曲霉毒素的降解效率。结果表明:地衣芽孢杆菌的上清液降解活性最高,确定有效活性成分存在于上清液中;对上清液进行高温和蛋白酶K变性处理后,降解活性显著降低,初步判定降解黄曲霉毒素B_1的有效活性成分是一种胞外酶。进一步利用高效液色谱对黄曲霉毒素B_1的降解产物进行了研究,并检测到降解产物P1和P2。
The chemical structure of aflatoxins(AFs) is very similar to coumarin. It has strong toxicity,carcinogenicity,and is an unavoidable pollutant in feed and foods. It is urgent to develop effective and environmentally friendly detoxification methods for degrading aflatoxins. In order to isolate bacteria with AFs degradation activity,screening was carried out using coumarin,a structural analogue of AFs,as the sole carbon source. As a result,one strain(HH-1) with high degradation rate(91.86%) was obtained. 16 S r DNA gene sequencing confirmed it as a strain of Bacillus licheniformis. Broth culture of HH-1 were divided into different components including culture supernatant and bacterial cell. The results indicated that the activity of aflatoxins degradation mainly existed in the culture supernatant of strain HH-1 rather than in bacterial cells. High temperature and protease K treatments suggested that its degradation activity was largely due to the action of extracellular enzymes. The degradation products P_1 and P_2 of AFs were also detected by high performance liquid chromatography.
The chemical structure of aflatoxins(AFs) is very similar to coumarin. It has strong toxicity,carcinogenicity,and is an unavoidable pollutant in feed and foods. It is urgent to develop effective and environmentally friendly detoxification methods for degrading aflatoxins. In order to isolate bacteria with AFs degradation activity,screening was carried out using coumarin,a structural analogue of AFs,as the sole carbon source. As a result,one strain(HH-1) with high degradation rate(91.86%) was obtained. 16 S r DNA gene sequencing confirmed it as a strain of Bacillus licheniformis. Broth culture of HH-1 were divided into different components including culture supernatant and bacterial cell. The results indicated that the activity of aflatoxins degradation mainly existed in the culture supernatant of strain HH-1 rather than in bacterial cells. High temperature and protease K treatments suggested that its degradation activity was largely due to the action of extracellular enzymes. The degradation products P_1 and P_2 of AFs were also detected by high performance liquid chromatography.
引文
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[2] HRUSKA Z,YAO H,KINCAID R,et al. Fluorescence excitation-emission features of aflatoxin and related secondary metabolites and their application for rapid detection of mycotoxins[J].Food and Bioprocess Technology,2014,7(4):1195-1201.
[3] GROSS-STEINMEYER K,EATON D L. Dietary modulation of the biotransformation and genotoxicity of aflatoxin B1[J]. Toxicology,2012,299(2/3):69-79.
[4] INOUE T,NAGATOMI Y,UYAMA A,et al.Degradation of aflatoxin B1during the fermentation of alcoholic beverages[J]. Toxins,2013,5(7):1219-1229.
[5]岳清华,赵仁勇,侯赛,等.几种降解玉米中黄曲霉毒素方法的比较[J].粮食与饲料工业,2015,10(6):15-19.
[6] HWANG J H,LEE K G. Reduction of aflatoxin B1contamination in wheat by various cooking treatments[J]. Food Chemistry,2006,98:71-75.
[7]赵仁勇,宋斌.玉米赤霉烯酮生物脱毒研究进展[J].河南工业大学学报(自然科学版),2018,39(2):113-121.
[8] HERZALLAH S,ALSHAWABKEH K,FATAFTAH A A L. Aflatoxin decontamination of artificially contaminated feeds by sunlight,γ-Ra diation,and microwave heating[J]. Journal of Applied Poultry Research,2008,17(4):515-521.
[9] DIAZ D E,HAGLER JR W M,HOPKINS B A,et al. Aflatoxin binders I:in vitro binding assay for aflatoxin B1by several potential sequestering agents[J]. Mycopathologia,2003,156(3):223-226.
[10]张勇,朱宝根.二氧化氯对霉变玉米黄曲霉毒素B1脱毒效果的研究[J].食品科学,2001,22(10):68-70.
[11] M魪NDEZ-ALBORES A,A RAMBULA-VILLA G,LOARCA-PI譙A M G F,et al. Safety and efficacy evaluation of aqueous citric acid to degrade B-aflatoxins in maize[J]. Food and Chemical Toxicology, 2005,43(2):233-238.
[12] TENIOLA O D,ADDO P A,BROST I M,et al. Degradation of aflatoxin B1by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov.DSM44556 T[J]. International Journal of Food Microbiology,2005,105(2):111-117.
[13] FARZANEH M,SHI Z Q,GHASSEMPOUR A,et al. Aflatoxin B1degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran[J]. Food Control,2012,23(1):100-106.
[14]雷元培,赵丽红,马秋刚,等.降解黄曲霉毒素枯草芽孢杆菌的解毒性、抗菌性及抗逆性研究[J].饲料工业,2011,32(24):23-27.
[15]布坎南R E,吉本斯N E.伯杰氏细菌鉴定手册[M].北京:科学出版社,1984.
[16]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001.
[17]李俊霞,梁志宏,关舒,等.黄曲霉毒素B1降解菌株的筛选及鉴定[J].中国农业科学,2008,41(5):1459-1463.
[18]刘桂君,朱婷婷,刘红霞,等.清香大曲及酒醅中地衣芽孢杆菌的分离鉴定[J].酿酒科技,2010(1):31-35.
[19] LEE L S,DUNN J J,DELUCCA A J,et al.Role of lactone ring of aflatoxin B1in toxicity and mutagenicity[J]. Experientia,1981,37(1):16-17.
[20] LIU D L,YAO D S,LIANG R,et al. Detoxification of aflatoxin B1by enzymes isolated from Armillariella tabescens[J]. Food and Chemical Toxicology,1998,36(7):563-574.