一株高效降解玉米赤霉烯酮的耐酸耐高温枯草芽孢杆菌的研究
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摘要
为寻求更高效、实用的生物脱毒制剂,提高其应用范围,通过采集酸性、高温的温泉环境样品,筛选获得1株高效降解玉米赤霉烯酮(ZEN)的细菌菌株Y-33,并对该菌株生长和降解特性进行分析。结果表明,菌株Y-33在50℃、pH值5.0条件下孵育36 h,菌液对2μg·mL~(-1) ZEN的降解率高达93.79%,对20μg·mL~(-1) ZEN的降解率也达82.40%。从形态、生理生化特性及16S rDNA序列对菌株Y-33进行分析,初步鉴定为枯草芽孢杆菌(Bacillus subtilis)。该菌株发酵上清液在28℃条件下对ZEN降解率为62.02%。金属离子Mn~(2+)能明显增强菌株Y-33发酵上清液对ZEN的降解能力,降解率达81.17%,而Zn~(2+)严重抑制了Y-33发酵上清液对ZEN的降解能力,降解率仅为5.42%。本研究结果为ZEN解毒菌剂的开发与应用奠定了理论基础。
To seek more efficient and practical biological detoxification strain to improve the application scope, we collected samples from acid and high temperature environment. One strain of bacteria named Y-33, was isolated for efficient detoxification zearalenone(ZEN),. Y-33 strain was incubated at 50℃ and pH value 5.0 for 36 hours, after which the degradation rate of bacterial solution to 2 μg·mL~(-1) ZEN was as high as 93.79%, and up to 82.40% to 20 μg·mL~(-1) ZEN. The strain Y-33 was initially identified as Bacillus subtilis with the analysis of its morphological, physiological and biochemical characteristics and 16 S rDNA sequences. The degradation rate in fermentation supernatant of the strain′s was 62.02% at 28℃. Metal ion Mn~(2+) could significanty enhance the degradation ability of strain Y-33 culture supernatant to ZEN with the degradation rate being 81.17%, while Zn~(2+) severely inhibited the degradation capability of ZEN by Y-33 culture supernatant to only 5.42%. The results of this study lay the theoretical foundation for the development and application of ZEN detoxifying microbes.
To seek more efficient and practical biological detoxification strain to improve the application scope, we collected samples from acid and high temperature environment. One strain of bacteria named Y-33, was isolated for efficient detoxification zearalenone(ZEN),. Y-33 strain was incubated at 50℃ and pH value 5.0 for 36 hours, after which the degradation rate of bacterial solution to 2 μg·mL~(-1) ZEN was as high as 93.79%, and up to 82.40% to 20 μg·mL~(-1) ZEN. The strain Y-33 was initially identified as Bacillus subtilis with the analysis of its morphological, physiological and biochemical characteristics and 16 S rDNA sequences. The degradation rate in fermentation supernatant of the strain′s was 62.02% at 28℃. Metal ion Mn~(2+) could significanty enhance the degradation ability of strain Y-33 culture supernatant to ZEN with the degradation rate being 81.17%, while Zn~(2+) severely inhibited the degradation capability of ZEN by Y-33 culture supernatant to only 5.42%. The results of this study lay the theoretical foundation for the development and application of ZEN detoxifying microbes.
引文
[1] 唐语谦,钟凤,陈艺,史成超,李丹,吴晖.玉米赤霉烯酮生物脱毒及关键酶作用机理的研究进展[J].现代食品科技,2013,29(7):1742-1746
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[19] Takahashi-Ando N,Kimura M,Kakeya H,Osada H,Yamaguchi Ⅰ.A novel lactonohydrolase responsible for the detoxification of zearalenone:enzyme purification and gene cloning[J].Biochemical Journal,2002,365(Pt1):1-6
[20] Tan H,Zhang Z M,Hu Y C,Wu L,Liao F.Isolation and characterization of Pseudomonas otitidis TH - N1 capable of degrading Zearalenone [J].Food Control,2015,47:285-290
[21] Yu Y,Qiu L,Wu H,Tang Y Q,Yu Y G,Li X F,Liu D M.Degradation of zearalenone by the extracellular extracts of Acinetobacter sp.SM04 liquid cultures [J].Biodegradation,2010,22(3):613-622
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[23] Lei Y P,Zhao L H,Ma Q G,Zhang J Y,Zhou T,Gao C Q,Ji C.Degradation of zearalenone in seine feed and feed ingredients by Bacillus subtilis ANSB01G [J].World Mycotoxin Journal,2014,7(2):143-151
[24] 龙淼,李鹏,朱连勤,王哲.微生物降解玉米赤霉烯酮毒素及其机制[J].动物医学进展,2011,32(11):116-119
[25] Tinyiro S E,Yao W,Sun X,Wokadala C,Wang S.Scavenging of zearalenone by bacillus strains in vitro [J].Research Journal Microbiology,2011,6(3):304-309
[26] Tinyiro S E,Wokadala C,Xu D,Yao W.Adsorption and degradation of zearalenone by bacillus strains [J].Folia Microbiologica,2011,56(4):321-327
[27] Yi P J,Pai C K,Liu J R.Isolation and characterization of a Bacillus licheniformis strain capable of degrading zearalenone [J].World Journal of Microbiol & Biotechnol,2011,27(5):1035-1043
[28] Urraca J L,Beni to-Peň a E,Pérez-Conde C,Moreno-Bondi M C,Pestka J J.Analysis of earalenone in cereal and swine feed samples using an automated flow-through immunosensor[J].Agricultural and Food Chemistry,2005,53(9):33-38
[29] 侯月娥,姜瑞丽,李旭宁,田书会.饲料和原料中玉米赤霉烯酮的危害及检测方法探讨[J].饲料与营养,2014(3):30-33
[30] Sun Y N,Hu X F,Zhang Y,Yang J,Wang F.Development of an Immunochromatographic strip test for the rapid detection of zearalenone in corn[J].Journal of Agricultural and Food Chemistry,2014,62(46):11116-11121
[31] 龚珊,党献民,任正东,刘旭,李荛.C18 固相萃取柱净化-超高效液相色谱法检测谷物中玉米赤霉烯酮[J].粮食与饲料工业,2015(6):60-63
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[35] Tamura K,Dudley J,Nei M,Kumar S.MEGA4:molecular evolutionary genetics analysis (MEGA) software version 4.0[J].Molecular Biology Evolution,2007,24(8):1596-1599
[36] Westley B,Rochefort H.A secreted glycoprotein induced by estrogen in human breast cancer cell lines [J].Cell,1980,20(2):353-362
[37] Guan S,Zhao L,Ma Q,Zhou T,Wang N.In vitro efficacy of Myxococcus fulvus ANSM068 to biotransform aflatoxin B1[J].Intternational Journal of Molecular Sciences,2010,11(10):4063-4079
[38] 冯胜军,吴丽丹,孙万邦,冯胜军,肖政,米娜,刘仿,陈群.不同培养基上空肠弯曲菌生长特性及外膜蛋白表达差异的初步研究[J].南方医科大学学报,2009,29(11):2279-2283
[39] Altalhi A D,EI-Deeb B.Localization of zearalenone detoxification gene(s) in pZEA-1 plasmid of Pseudomorias putida ZEA-1 and expressed in Escherichia coli [J].Journal of Harardous Materials,2009,161(2/3):1166-1172
[40] 张初署,孙杰,毕洁,于丽娜,张建成,胡玉忠,赵善仓.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
[41] 董曼佳,杨其亚,孙伟,张红印,李超兰.拮抗酵母菌控制玉米赤霉烯酮的研究进展[J].食品科学,2016,37(1):230-234
[42] Luiz K,Luís A,Kelly K,Carlos A R,Lilia C,Armando Ⅴ.Zearalenone and its derivatives α-zearalenol and β-zearalenol decontamination by Saccharomyces cerevisiae strains isolated from bovine forage[J].Toxins,2015,7(8):3297-3308
[43] Zhang H,Dong M,Yang Q,Apaliya M T,Li T.Biodegradation of zearalenone by Saccharomyces cerevisiae:Possible involvement of ZEN responsive proteins of the yeast[J].Journal of Proteomics,2016,143:416-423
[44] Kakeya H,Takahashi-Ando N,Kimura M,Onose R,Yamaguchi Ⅰ,Osada H.Biotransform of the mycotoxin,zearalenone,to a non-esrtogenic compound by a fungal strain of Clonostachys sp.[J].Biosci Biotechnol Biochem,2002,66(12):2723-2726
[45] Cho K J,Kang J S,Cho W T,Lee C H,Ha J K,Song K B.In vitro degradation of zearalnone by Bacillus subtilis [J].Biotechnolgy Letters,2010,32(12):1921-1924
[46] 刘大岭,姚冬生,冼钰茵,孙志轩,谢春芳,梁玉强,梁钟,邵伟琪.一种分解玉米赤霉烯酮和木聚糖的枯草芽孢杆菌及其应用:中国,103667103 [P].2016-01-20
[2] 陈继发,张佳鑫,曲湘勇,彭豫东.玉米赤霉烯酮对猪的毒性作用及其生物降解研究进展[J].中国饲料,2015(19):30-34,41
[3] 温琦,苏从毅,何武顺,李爱科,王四维.饲料中真菌毒素的危害与限量[J].饲料广角,2014(5):32-36
[4] Zaki M,Midany S A E,Shaheen H,Rizzi L.Mycotoxins in animals:Occurrence,effects,prevention and management[J].Journal of Toxicology and Environmental Health Sciences,2012,4(1):13-28
[5] Warth B,Sulyok M,Berthiller F,Schuhmacher R,Krska R.New insights into the human metabolism of the Fusarium mycotoxins deoxynivalenol and zearalenone[J].Toxicology Letters,2013,220(1):88-94
[6] 董双,何剑斌,张燚,龙淼.玉米赤霉烯酮对雄性小鼠生殖系统的毒性作用[J].畜牧与兽医,2016(6):25-29
[7] 马传国,王英丹.玉米赤霉烯酮污染状况及毒性的研究进展[J].河南工业大学学报(自然科学版),2017,38(2):122-127
[8] 戎晓平,赵丽红,计成,马秋刚,王馨悦,周建川,刘尧君,张丽元.我国部分地区饲料原料及配合饲料玉米赤霉烯酮污染情况调研[J].中国畜牧杂志,2015,51(22):20-24
[9] 刘盼,蔡俊.玉米赤霉烯酮生物脱毒与降解的研究进展[J].中国酿造,2017,36(2):1-5
[10] 史競,汪洋,鞠星,宾石玉,张晓琳.玉米赤霉烯酮生物降解研究进展[J].中国粮油学报,2013,28(6):111-114
[11] 路子显,伍松陵孙长坡.玉米赤霉烯酮生物合成和降解的研究进展[J].中国生物工程杂志,2011,31(2):116-122
[12] Jouany J P,Jiang Y,Ogunade Ⅰ M,Kim D H,Adesogan A T.Methors for preventing,decontaminating and minimizing the toxicity of mycotoxins in feeds [J].Animal Feed Science and Technology,2007,137(3):342-362
[13] 李溪,王金荣,赵银丽,苏兰利,朱雪飞,陈培英.饲料中玉米赤霉烯酮脱毒技术研究[J].饲料研究,2015(13):16-21
[14] Bullerman L B,Bianchini A.Stability of myeotoxin during food processing [J].International Journal of Food Microbiology,2007,119(1/2):140-146
[15] Abd Alla E S.Zearalenone:Incidence,toxigenic fungi and chemical decontamination in Egyptian cereals [J].Food/Nahrung,1998,41(6):362-365
[16] 李顺意,于秋香,向腊,周玉玲,张桂敏.真菌毒素玉米赤霉烯酮生物降解的研究进展[J].生物工程学报,2018,34(4):489-500
[17] 熊凯华,程波财,胡威,汪孟娟,叶若松,魏华.玉米赤霉烯酮降解的研究进展[J].中国粮油学报,2010,25(1):125-138
[18] 赵琳,张瑞英,兰静,金海涛,任红波,孙向东.γ-射线降解玉米粉中玉米赤霉烯酮的效果及降解动力学研究[J].食品工业科技,2016,37(7):56-60
[19] Takahashi-Ando N,Kimura M,Kakeya H,Osada H,Yamaguchi Ⅰ.A novel lactonohydrolase responsible for the detoxification of zearalenone:enzyme purification and gene cloning[J].Biochemical Journal,2002,365(Pt1):1-6
[20] Tan H,Zhang Z M,Hu Y C,Wu L,Liao F.Isolation and characterization of Pseudomonas otitidis TH - N1 capable of degrading Zearalenone [J].Food Control,2015,47:285-290
[21] Yu Y,Qiu L,Wu H,Tang Y Q,Yu Y G,Li X F,Liu D M.Degradation of zearalenone by the extracellular extracts of Acinetobacter sp.SM04 liquid cultures [J].Biodegradation,2010,22(3):613-622
[22] Abdulla D,El-Deeb B.Localization of zearalenone detoxification gene(s) in pZEA-1 plasmid of Pseudomonas putida ZEA-1 and expressed in Escherichia coli[J].Journal of Hazardous Materials,2009,161(2/3):1166-1172
[23] Lei Y P,Zhao L H,Ma Q G,Zhang J Y,Zhou T,Gao C Q,Ji C.Degradation of zearalenone in seine feed and feed ingredients by Bacillus subtilis ANSB01G [J].World Mycotoxin Journal,2014,7(2):143-151
[24] 龙淼,李鹏,朱连勤,王哲.微生物降解玉米赤霉烯酮毒素及其机制[J].动物医学进展,2011,32(11):116-119
[25] Tinyiro S E,Yao W,Sun X,Wokadala C,Wang S.Scavenging of zearalenone by bacillus strains in vitro [J].Research Journal Microbiology,2011,6(3):304-309
[26] Tinyiro S E,Wokadala C,Xu D,Yao W.Adsorption and degradation of zearalenone by bacillus strains [J].Folia Microbiologica,2011,56(4):321-327
[27] Yi P J,Pai C K,Liu J R.Isolation and characterization of a Bacillus licheniformis strain capable of degrading zearalenone [J].World Journal of Microbiol & Biotechnol,2011,27(5):1035-1043
[28] Urraca J L,Beni to-Peň a E,Pérez-Conde C,Moreno-Bondi M C,Pestka J J.Analysis of earalenone in cereal and swine feed samples using an automated flow-through immunosensor[J].Agricultural and Food Chemistry,2005,53(9):33-38
[29] 侯月娥,姜瑞丽,李旭宁,田书会.饲料和原料中玉米赤霉烯酮的危害及检测方法探讨[J].饲料与营养,2014(3):30-33
[30] Sun Y N,Hu X F,Zhang Y,Yang J,Wang F.Development of an Immunochromatographic strip test for the rapid detection of zearalenone in corn[J].Journal of Agricultural and Food Chemistry,2014,62(46):11116-11121
[31] 龚珊,党献民,任正东,刘旭,李荛.C18 固相萃取柱净化-超高效液相色谱法检测谷物中玉米赤霉烯酮[J].粮食与饲料工业,2015(6):60-63
[32] R E,N.E.伯杰氏系统细菌学手册[M].北京:科学出版社,1984
[33] 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
[34] Chun J,Lee J H,Jung Y,Kim M,Kim S,Kim B K,Lim Y W.EzTaxon:A web-based tool for the identification of prokaryotes based on 16S ribosomal RNA gene sequences[J].International Journal of Systematic and Evolution Microbiology,2007,57(10):2259-2261
[35] Tamura K,Dudley J,Nei M,Kumar S.MEGA4:molecular evolutionary genetics analysis (MEGA) software version 4.0[J].Molecular Biology Evolution,2007,24(8):1596-1599
[36] Westley B,Rochefort H.A secreted glycoprotein induced by estrogen in human breast cancer cell lines [J].Cell,1980,20(2):353-362
[37] Guan S,Zhao L,Ma Q,Zhou T,Wang N.In vitro efficacy of Myxococcus fulvus ANSM068 to biotransform aflatoxin B1[J].Intternational Journal of Molecular Sciences,2010,11(10):4063-4079
[38] 冯胜军,吴丽丹,孙万邦,冯胜军,肖政,米娜,刘仿,陈群.不同培养基上空肠弯曲菌生长特性及外膜蛋白表达差异的初步研究[J].南方医科大学学报,2009,29(11):2279-2283
[39] Altalhi A D,EI-Deeb B.Localization of zearalenone detoxification gene(s) in pZEA-1 plasmid of Pseudomorias putida ZEA-1 and expressed in Escherichia coli [J].Journal of Harardous Materials,2009,161(2/3):1166-1172
[40] 张初署,孙杰,毕洁,于丽娜,张建成,胡玉忠,赵善仓.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
[41] 董曼佳,杨其亚,孙伟,张红印,李超兰.拮抗酵母菌控制玉米赤霉烯酮的研究进展[J].食品科学,2016,37(1):230-234
[42] Luiz K,Luís A,Kelly K,Carlos A R,Lilia C,Armando Ⅴ.Zearalenone and its derivatives α-zearalenol and β-zearalenol decontamination by Saccharomyces cerevisiae strains isolated from bovine forage[J].Toxins,2015,7(8):3297-3308
[43] Zhang H,Dong M,Yang Q,Apaliya M T,Li T.Biodegradation of zearalenone by Saccharomyces cerevisiae:Possible involvement of ZEN responsive proteins of the yeast[J].Journal of Proteomics,2016,143:416-423
[44] Kakeya H,Takahashi-Ando N,Kimura M,Onose R,Yamaguchi Ⅰ,Osada H.Biotransform of the mycotoxin,zearalenone,to a non-esrtogenic compound by a fungal strain of Clonostachys sp.[J].Biosci Biotechnol Biochem,2002,66(12):2723-2726
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