一株黄曲霉毒素B_1降解菌的筛选及鉴定
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摘要
以香豆素为唯一碳源进行AFB1降解菌的初筛,从青岛土壤中筛选出高效降解AFB1的菌株A6,分离该菌的上清液、菌悬液、胞内液并分析各组分降解AFB_1特征,通过形态学、生理生化和16S rRNA基因对该菌株进行鉴定。研究结果表明,菌株A6能高效降解AFB1,其胞外上清液、菌悬液和胞内液能分别降解90.6%、19.6%和12.8%的AFB_1,表明起降解的活性物质主要位于胞外液。在平板上该菌落呈现乳白色,具有皱褶,革兰氏染色呈阳性;能利用葡萄糖、阿拉伯糖、乳糖等碳源; 16S rRNA基因分析表明该菌与贝莱斯芽孢杆菌CR-502~T(Bacillus velezensis)相似度为99.64%。综合形态学特征、生理生化特征和16S rRNA基因分析结果,鉴定菌株A6为贝莱斯芽孢杆菌(Bacillus velezensis),命名为Bacillus velezensis A6。本研究结果为深入研究该菌的降解机理奠定了基础。
The degradation bacteria were isolated from the soil in Qingdao by screening with coumarin as the only carbon source,and the highly efficient strain A6 was further identified.The supernate,bacterium suspension and intracellular fluid of the strain A6 were separated and the degradation characteristics of AFB_1 were analyzed.The strain was identified by morphological characteristics,physiological,biochemical characteristics and 16 S rRNA gene.The results indicated that the strain A6 could efficiently degrade AFB_1.The supernate,bacterium suspension and intracellular fluid could degrade 90.6%,19.6%and 12.8% of AFB_1,suggesting that the degradation activity of strain A6 was located in extracellular fluid.On the plate,the strain colony was milky white with wrinkles and gram stain was positive.The strain could assimilate glucose,arabinose,lactose and other carbon sources.Analysis of the 16 S rRNA gene showed that the similarity between strain A6 and Bacillus velezensis CR-502~T was 99.64%.Based on the morphology,physiological and biochemical characteristics,and the analysis of 16 S rRNA gene sequence homology,the strain A6 was identified as Bacillus velezensis.The results provided a foundation for further study on the degradation mechanism of this bacterium.
The degradation bacteria were isolated from the soil in Qingdao by screening with coumarin as the only carbon source,and the highly efficient strain A6 was further identified.The supernate,bacterium suspension and intracellular fluid of the strain A6 were separated and the degradation characteristics of AFB_1 were analyzed.The strain was identified by morphological characteristics,physiological,biochemical characteristics and 16 S rRNA gene.The results indicated that the strain A6 could efficiently degrade AFB_1.The supernate,bacterium suspension and intracellular fluid could degrade 90.6%,19.6%and 12.8% of AFB_1,suggesting that the degradation activity of strain A6 was located in extracellular fluid.On the plate,the strain colony was milky white with wrinkles and gram stain was positive.The strain could assimilate glucose,arabinose,lactose and other carbon sources.Analysis of the 16 S rRNA gene showed that the similarity between strain A6 and Bacillus velezensis CR-502~T was 99.64%.Based on the morphology,physiological and biochemical characteristics,and the analysis of 16 S rRNA gene sequence homology,the strain A6 was identified as Bacillus velezensis.The results provided a foundation for further study on the degradation mechanism of this bacterium.
引文
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[20]孙玲玉,李超,郝海玉,等.泰山枯草芽孢杆菌的分离鉴定及其对黄曲霉毒素B1的降解研究[J].中国畜牧兽医,2014,41:246-250.
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[22]Hwang,C Y,Zhang G I,Kang S H,et al.Pseudomonas pelagia sp.nov.,isolated from a culture of the Antarctic green alga Pyramimonas gelidicola[J].International Journal of Systematic and Evolutionary Microbiology,2009,59:3019-3024.
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[26]Hernandez-Mendoza A,Garcia H S,Steele J L.Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1[J].Food and Chemical Toxicology,2009,47(6):1064-1068.
[27]El-Nezami H,Kankaanpaa P,Salminen S,et al.Ability of dairy strains of lactic acid vacteria to bind a common food carcinogen,aflatoxin B1[J].Food and Chemical Toxicology,1998,36(4):321-326.
[28]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.
[2]Perrone G,Susca A,Cozzi G,et al.Biodiversity of Aspergillus species in some important agricultural products[J].Studies in Mycology,2007,59(7):53-66.
[3]Steyn P S.Mycotoxins,general view,chemistry and structure[J].Toxicology Letters,1995,82(83):843-851.
[4]Xu L,Eisa-Ahmed M F,Sangare L,et al.Novel aflatoxindegrading enzymes from Bacillus shackletonii L7[J].Toxins,2017,9(1):36.
[5]吉小凤,张巧艳,李文均,等.黄曲霉毒素B1脱毒菌株LAB-10的分离、鉴定及降解能力分析[J].微生物学通报,2012,39(8):1094-1101.
[6]图彩虹,秦文,胡欣洁,等.一株黄曲霉拮抗细菌的分离筛选及鉴定[J].食品工业科技,2011,32(4):182-188.
[7]Ono E Y,Ono M A,Funo F Y,et al.Evaluation of fumonosinaflatoxin co-occurrence in Brazilian corn hybrids by ELISA[J].Food Additives and Contaminants,2001,18(8):719-729.
[8]王德培,李可乐,王亚军,等.解淀粉芽孢杆菌BI2抗真菌活性物质的分离纯化及特性分析[J].食品工业科技,2013,34(9):78-81.
[9]Gowda N K S,Suganthhi R U,MaLathi V,et al.Efficacy of heat treatment and sun drying of aflatoxin-contaminated feed for reducing the harmful biological effects in sheep[J].Animal Feed Science and Technilogy,2007,133(1-2):167-175.
[10]Desheng Q,Fan L,Yanhu Y,et al.Adsorption of aflatoxin B1on montmorillonite[J].Poultry Science,2005,85:959-961.
[11]关心,何建斌,董双,等.黄曲霉毒素B1高效降解菌株的筛选鉴定及其降解[J].华中农业大学学报,2016,35(2):90-96.
[12]Womack E D,Brown A E,Sparks D L.A recent review of non-biological remediation of aflatoxin-contaminated crops[J].Society of Chemical Industry,2014,94(9):1706-1714.
[13]张初署,孙杰,毕洁,等.食用菌SJ-1漆酶酶学性质及降解黄曲霉毒素B1的研究[J].核农学报,2017,31(7):1317-1322.
[14]杨文华,李海星,刘晓华,等.黄曲霉毒素B1降解酶的分离纯化及其酶学特征[J].食品科学,2014,35(19):164-168.
[15]Fan Y,Liu L T,Zhao L H,et al.Influence of Bacillus subtilis ANSB060 on growth,digestive enzyme and aflatoxin residue in Yellow River carp fed diets contaminated with aflatoxin B1[J].Food and Chemical Toxicology,2018,113:108-114.
[16]Sangare L,Zhao Y J,Elodie-Folly Y M,et al.Aflatoxin B1degradation by a Pseudomonas strain[J].Toxins,2014,6:3028-3040.
[17]Liu D L,Yao D S,Liang Y Q,et al.Production,purification,and characterization of an intracellular aflatoxin-detoxifizyme from Armillariella tabescens(E20)[J].Food and Chemical Toxicology,2001,39:461-466.
[18]Guan S,Ji C,Zhou T,et al.Aflatoxin B1degradation by Stenotrophomonas maltophilia and other microbes selected using coumarin medium[J].International Journal of Molecular Sciences,2008,9:1489-1503.
[19]王明清,于丽娜,张初署,等.黄曲霉毒素B1降解菌的分离、鉴定及其降解能力研究[J].花生学报,2018,47(2):1-5.
[20]孙玲玉,李超,郝海玉,等.泰山枯草芽孢杆菌的分离鉴定及其对黄曲霉毒素B1的降解研究[J].中国畜牧兽医,2014,41:246-250.
[21]Wang,M Q,Sun L.Pseudomonas oceani sp.nov.,isolated from deep seawater[J].International Journal of Systematic and Evolutionary Microbiology,2016,66(10):4250-4255.
[22]Hwang,C Y,Zhang G I,Kang S H,et al.Pseudomonas pelagia sp.nov.,isolated from a culture of the Antarctic green alga Pyramimonas gelidicola[J].International Journal of Systematic and Evolutionary Microbiology,2009,59:3019-3024.
[23]Kim K H,Roh S W,Chang H W,et al.Pseudomoni sabulinigri sp.nov.,isolated from black beach sand[J].International Journal of Systematic and Evolutionary Microbiology,2009,59:38-41.
[24]Frank J A,Reich C I,Sharma S,et al.Critical evaluation of two primers commonly used for amplification of bacterial 16Sr RNA genes[J].Applied and Environmental Microbiology,2008,74(8):2461-2470.
[25]Ruiz-Garía C,Béjar V,Martínez-Checa F,et al.Bacillus velezensis sp.nov.,a surfactant-production bacterium isolated from the river Vélez in Málaga,southern Spain[J].International Journal of Systematic and Evolutionary Microbiology,2005,55:191-195.
[26]Hernandez-Mendoza A,Garcia H S,Steele J L.Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1[J].Food and Chemical Toxicology,2009,47(6):1064-1068.
[27]El-Nezami H,Kankaanpaa P,Salminen S,et al.Ability of dairy strains of lactic acid vacteria to bind a common food carcinogen,aflatoxin B1[J].Food and Chemical Toxicology,1998,36(4):321-326.
[28]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.