黄曲霉毒素B1降解菌的筛选及在玉米贮藏中的应用
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摘要
【目的】本研究分离筛选出一株对黄曲霉既有抑制作用又能降解其毒素的拮抗细菌菌株,并将其应用于玉米中的黄曲霉污染防治研究。【方法】试验通过平板筛选法结合玉米活体筛选法对黄曲霉毒素B1 (AFB1)的拮抗细菌进行初筛,以AFB1的降解率和抑制率为指标进行复筛。【结果】分离到的菌株对黄曲霉菌的抑制率为79.20%,对1μg/mL的黄曲霉毒素B1的降解率为68.39%。贮藏期玉米含水量在15%–30%时,该菌株对黄曲霉污染的抑制率与玉米含水量成反比,即玉米含水量在15%时其抑制率达92.46%,玉米含水量在30%时其抑制率为19.41%。玉米含水量在28%时,菌株对黄曲霉污染玉米的防治效果达到36.39%。【结论】筛选出的拮抗菌株为枯草芽孢杆菌,该菌株不仅对黄曲霉菌有抑制作用,而且能减少AFB1对玉米的污染。
[Objective] We isolated bacterial strain that can antagonize the aflatoxin B1(AFB1) producing fungus Aspergillus flavus in maize seed. [Methods] The plate method and in vivo screening were performed for samples from various sources. The positive bacterial strains were screened by their ability to degrade AFB1 in culture media. [Results] The inhibitory rate of bacterial strain to Aspergillus flavus was 79.2% and its degradation rate was 68.39% in 1 μg/mL AFB1. During storage period, the inhibition rate of the bacterial strain to Aspergillus flavus was inversely proportional to the water content of maize. When the water content in the maize was 15%, 92.46% inhibition rate was recorded, while the inhibition rate of 19.41% was noted with 30% water content in maize. When the maize water content was 28%, the control effect on Aspergillus flavus was 36.39%. [Conclusion] An AFB1-degrading strain not only could inhibit the growth of Aspergillus flavus, but also may control the contamination of AFB1 to maize.
[Objective] We isolated bacterial strain that can antagonize the aflatoxin B1(AFB1) producing fungus Aspergillus flavus in maize seed. [Methods] The plate method and in vivo screening were performed for samples from various sources. The positive bacterial strains were screened by their ability to degrade AFB1 in culture media. [Results] The inhibitory rate of bacterial strain to Aspergillus flavus was 79.2% and its degradation rate was 68.39% in 1 μg/mL AFB1. During storage period, the inhibition rate of the bacterial strain to Aspergillus flavus was inversely proportional to the water content of maize. When the water content in the maize was 15%, 92.46% inhibition rate was recorded, while the inhibition rate of 19.41% was noted with 30% water content in maize. When the maize water content was 28%, the control effect on Aspergillus flavus was 36.39%. [Conclusion] An AFB1-degrading strain not only could inhibit the growth of Aspergillus flavus, but also may control the contamination of AFB1 to maize.
引文
[1]Wang W,Li G,Wei YX.Research status of aflatoxin contamination in China.Journal of Anhui Agricultural Sciences,2015,43(18):308-309.(in Chinese)王雯,李岗,魏云潇.我国食品中黄曲霉毒素污染现状的研究.安徽农业科学,2015,43(18):308-309.
[2]Liu FZ,Sun HM,Lian XH,Li CF,Cong QX,Zhang TT,Yin XL,Gu W.Analysis of contamination status of aflatoxin B1 in feed and feed ingredients in 2014.Guangdong Feed,2015,24(3):45-46.(in Chinese)刘凤芝,孙合美,廉新慧,李春凤,丛秋霞,张甜甜,尹训兰,谷巍.2014年饲料及饲料原料中黄曲霉毒素B1污染状况的分析.广东饲料,2015,24(3):45-46.
[3]Li M,Qin RF,Chen DH.The effect of aflatoxin on livestock and poultry and the method of detoxification.Henan Journal of Animal Husbandry and Veterinary Medicine,2008,29(3):35-36.(in Chinese)李敏,秦锐峰,陈岱辉.黄曲霉毒素对畜禽的影响及去毒方法.河南畜牧兽医,2008,29(3):35-36.
[4]Farombi EO.Aflatoxin contamination of foods in developing countries:implications for hepatocellular carcinoma and chemopreventive strategies.African Journal of Biotechnology,2006,5(1):1-14.
[5]Gong YY,Hounsa A,Egal S,Turner PC,Sutcliffe AE,Hall AJ,Cardwell K,Wild CP.Postweaning exposure to aflatoxin results in impaired child growth:A longitudinal study in Benin,West Africa.Environmental Health Perspectives,2004,112(13):1334-1338.
[6]Zhen YZ,Liu GT.Research progress on carcinogenicity of mycotoxins in China.Acta Academiae Medicinae Henan,1983,(3):72-74.(in Chinese)甄应中,刘桂亭.国内真菌毒素致癌性的研究进展.河南医学院学报,1983,(3):72-74.
[7]Li JX,Ouyang SZ,Wang XP.Injuries of aflatoxin to animals and protective functions of selenium.Journal of Henan Vocation-Technical Teachers College,2003,31(2):50-54.(in Chinese)李敬玺,欧阳素贞,王学平.黄曲霉毒素对机体的损害与硒的保护作用.河南职业技术师范学院学报,2003,31(2):50-54.
[8]Wild CP,Gong YY.Mycotoxins and human disease:a largely ignored global health issue.Carcinogenesis,2010,31(1):71-82.
[9]中华人民共和国卫生部.食品中真菌毒素限量.北京:中国标准出版社,2011.
[10]Wang HL,Fan HT,Lu WY.Patent technology of aflatoxin detoxification method.Journal of Anhui Agricultural Sciences,2015,43(17):291-293.(in Chinese)王慧林,樊海涛,卢文英.黄曲霉毒素脱毒方法专利技术综述.安徽农业科学,2015,43(17):291-293.
[11]Liu DL,Yao DS,Huang BH,Xie CF,Liang YQ,Ma L.Characterization of immobilized aflatoxin-detoxizyme.Chinese Journal of Biotechnology,2003,19(5):603-607.(in Chinese)刘大岭,姚冬生,黄炳贺,谢春芳,梁郁强,马林.黄曲霉毒素解毒酶的固定化及其性质的研究.生物工程学报,2003,19(5):603-607.
[12]Chen ZH,Xu X,Li ZY,Cheng BJ.Study on the effect factors of degradation of aflatoxin B1 by Aspergillus niger.Food Research and Development,2015,36(12):110-113.(in Chinese)陈志辉,徐馨,李仲玉,程宝晶.黑曲霉菌降解黄曲霉毒素B1的影响因素的研究.食品研究与开发,2015,36(12):110-113.
[13]Smiley RD,Draughon FA.Preliminary evidence that degradation of aflatoxin B1 Flavobacterium aurantiacum is enzymatic.Journal of Food Protection,2000,63(3):415-418.
[14]Xu J,Ji R.The effect of lactic acid bacteria on the growth and production of fungi.Foreign Medical Hygienic Register,2001,28(4):237-240.(in Chinese)徐进,计融.乳酸菌对霉菌生长及产毒的影响.国外医学(卫生学分册),2001,28(4):237-240.
[15]Teniola OD,Addo PA,Brost IM,F?rber P,Jany KD,Alberts JF,van Zyl WH,Steyn PS,Holzapfel WH.Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp.nov.DSM44556T.International Journal of Food Microbiology,2005,105(2):111-117.
[16]Zhai CP,Kong Q,Guan B,Lv ZL,You Y,Gao YY,Yang YY.Statistical experimental methods for optimizing the cultivating conditions for Rhodococcus erythropolis.African Journal of Biotechnology,2011,10(42):8502-8510.
[17]Li JX,Liang ZH,Guan S,Wang X,Wang N,Ji C,Niu TG.Screening and identification of aflatoxin B1 degradation strains.Scientia Agricultura Sinica,2008,41(5):1459-1463.(in Chinese)李俊霞,梁志宏,关舒,汪晓,王宁,计成,牛天贵.黄曲霉毒素B1降解菌株的筛选及鉴定.中国农业科学,2008,41(5):1459-1463.
[18]Wang N,Ma QG,Ji C,Guan S,Niu TG.Screening of culture condition for aflatoxin B1 transformation enzyme from Myxococcus fulvus.Journal of China Agricultural University,2009,14(2):27-31.(in Chinese)王宁,马秋刚,计成,关舒,牛天贵.黏细菌降解黄曲霉毒素B1的产酶条件优化.中国农业大学学报,2009,14(2):27-31.
[19]Wang DP,Meng H,Guan XL,Luo XG.Identification of bacillus amyloliquefaciens BI2 and its antagonistic activity against Aspergillus flavus.Journal of Tianjin University of Science&Technology,2010,25(6):5-9.(in Chinese)王德培,孟慧,管叙龙,罗学刚.解淀粉芽孢杆菌BI2的鉴定及其对黄曲霉的抑制作用.天津科技大学学报,2010,25(6):5-9.
[20]Rao SQ,Chen SY,Gao L,Yin YQ,Yang ZQ,Fang WM.Screening,identification,and antifungal properties of a bacterial strain F1 with antagonistic activities against Aspergillus flavus.Modern Food Science and Technology,2015,31(12):99-105.(in Chinese)饶胜其,陈素雅,高璐,尹永祺,杨振泉,方维明.一株黄曲霉拮抗细菌F1的筛选、鉴定及其抑菌特性.现代食品科技,2015,31(12):99-105.
[21]Li CB,Li WM,Yang WH,Li HX,Liu XH,Cao YS.Isolation and characterisation of an aflatoxin B1-degrading bacterium.Acta Microbiologica Sinica,2012,52(9):1129-1136.(in Chinese)李超波,李文明,杨文华,李海星,刘晓华,曹郁生.黄曲霉毒素B1降解菌的分离鉴定及其降解特性.微生物学报,2012,52(9):1129-1136.
[22]韩愈杰.黄曲霉拮抗菌株的分离筛选及对黄曲霉毒素的降解效果.河北农业大学硕士学位论文,2015.
[23]Yue XY,Li ZG,Hao XZ,Xu J,Liu XD,Niu TG.Simulation of effect of main ecological factors on radial growth of Aspergillus flavus during storage period of corn.Transactions of the Chinese Society of Agricultural Engineering,2013,29(18):269-276.(in Chinese)岳晓禹,李自刚,郝修振,徐军,刘相东,牛天贵.主要生态因子对贮藏玉米中黄曲霉生长影响的模拟.农业工程学报,2013,29(18):269-276.
[24]Zhang P,He PF,Li ZX.Isolation and characterisation of aflatoxigen strains in corn.Southwest China Journal of Agricultural Sciences,2017,(30):146-150.(in Chinese)张佩,何鹏飞,李智新.玉米中产黄曲霉毒素菌株的分离与鉴定.西南农业学报(增刊),2017,(30):146-150.
[25]Shi JY,Ning H,Xu R,Gao ZX,Yu Y,Liu B,Ke YP,Sun Q.Inhibitory effect on the growth and aflatoxin-production of Aspergillus flavus by ClO2 and its application in corn of high water content.China Measurement&Test,2017,43(10):47-52.(in Chinese)史健阳,宁华,徐冉,高珍璇,余洋,刘保,柯永培,孙群.二氧化氯抑制黄曲霉菌生长和产毒能力及其在高水分玉米中的应用.中国测试,2017,43(10):47-52.
[2]Liu FZ,Sun HM,Lian XH,Li CF,Cong QX,Zhang TT,Yin XL,Gu W.Analysis of contamination status of aflatoxin B1 in feed and feed ingredients in 2014.Guangdong Feed,2015,24(3):45-46.(in Chinese)刘凤芝,孙合美,廉新慧,李春凤,丛秋霞,张甜甜,尹训兰,谷巍.2014年饲料及饲料原料中黄曲霉毒素B1污染状况的分析.广东饲料,2015,24(3):45-46.
[3]Li M,Qin RF,Chen DH.The effect of aflatoxin on livestock and poultry and the method of detoxification.Henan Journal of Animal Husbandry and Veterinary Medicine,2008,29(3):35-36.(in Chinese)李敏,秦锐峰,陈岱辉.黄曲霉毒素对畜禽的影响及去毒方法.河南畜牧兽医,2008,29(3):35-36.
[4]Farombi EO.Aflatoxin contamination of foods in developing countries:implications for hepatocellular carcinoma and chemopreventive strategies.African Journal of Biotechnology,2006,5(1):1-14.
[5]Gong YY,Hounsa A,Egal S,Turner PC,Sutcliffe AE,Hall AJ,Cardwell K,Wild CP.Postweaning exposure to aflatoxin results in impaired child growth:A longitudinal study in Benin,West Africa.Environmental Health Perspectives,2004,112(13):1334-1338.
[6]Zhen YZ,Liu GT.Research progress on carcinogenicity of mycotoxins in China.Acta Academiae Medicinae Henan,1983,(3):72-74.(in Chinese)甄应中,刘桂亭.国内真菌毒素致癌性的研究进展.河南医学院学报,1983,(3):72-74.
[7]Li JX,Ouyang SZ,Wang XP.Injuries of aflatoxin to animals and protective functions of selenium.Journal of Henan Vocation-Technical Teachers College,2003,31(2):50-54.(in Chinese)李敬玺,欧阳素贞,王学平.黄曲霉毒素对机体的损害与硒的保护作用.河南职业技术师范学院学报,2003,31(2):50-54.
[8]Wild CP,Gong YY.Mycotoxins and human disease:a largely ignored global health issue.Carcinogenesis,2010,31(1):71-82.
[9]中华人民共和国卫生部.食品中真菌毒素限量.北京:中国标准出版社,2011.
[10]Wang HL,Fan HT,Lu WY.Patent technology of aflatoxin detoxification method.Journal of Anhui Agricultural Sciences,2015,43(17):291-293.(in Chinese)王慧林,樊海涛,卢文英.黄曲霉毒素脱毒方法专利技术综述.安徽农业科学,2015,43(17):291-293.
[11]Liu DL,Yao DS,Huang BH,Xie CF,Liang YQ,Ma L.Characterization of immobilized aflatoxin-detoxizyme.Chinese Journal of Biotechnology,2003,19(5):603-607.(in Chinese)刘大岭,姚冬生,黄炳贺,谢春芳,梁郁强,马林.黄曲霉毒素解毒酶的固定化及其性质的研究.生物工程学报,2003,19(5):603-607.
[12]Chen ZH,Xu X,Li ZY,Cheng BJ.Study on the effect factors of degradation of aflatoxin B1 by Aspergillus niger.Food Research and Development,2015,36(12):110-113.(in Chinese)陈志辉,徐馨,李仲玉,程宝晶.黑曲霉菌降解黄曲霉毒素B1的影响因素的研究.食品研究与开发,2015,36(12):110-113.
[13]Smiley RD,Draughon FA.Preliminary evidence that degradation of aflatoxin B1 Flavobacterium aurantiacum is enzymatic.Journal of Food Protection,2000,63(3):415-418.
[14]Xu J,Ji R.The effect of lactic acid bacteria on the growth and production of fungi.Foreign Medical Hygienic Register,2001,28(4):237-240.(in Chinese)徐进,计融.乳酸菌对霉菌生长及产毒的影响.国外医学(卫生学分册),2001,28(4):237-240.
[15]Teniola OD,Addo PA,Brost IM,F?rber P,Jany KD,Alberts JF,van Zyl WH,Steyn PS,Holzapfel WH.Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp.nov.DSM44556T.International Journal of Food Microbiology,2005,105(2):111-117.
[16]Zhai CP,Kong Q,Guan B,Lv ZL,You Y,Gao YY,Yang YY.Statistical experimental methods for optimizing the cultivating conditions for Rhodococcus erythropolis.African Journal of Biotechnology,2011,10(42):8502-8510.
[17]Li JX,Liang ZH,Guan S,Wang X,Wang N,Ji C,Niu TG.Screening and identification of aflatoxin B1 degradation strains.Scientia Agricultura Sinica,2008,41(5):1459-1463.(in Chinese)李俊霞,梁志宏,关舒,汪晓,王宁,计成,牛天贵.黄曲霉毒素B1降解菌株的筛选及鉴定.中国农业科学,2008,41(5):1459-1463.
[18]Wang N,Ma QG,Ji C,Guan S,Niu TG.Screening of culture condition for aflatoxin B1 transformation enzyme from Myxococcus fulvus.Journal of China Agricultural University,2009,14(2):27-31.(in Chinese)王宁,马秋刚,计成,关舒,牛天贵.黏细菌降解黄曲霉毒素B1的产酶条件优化.中国农业大学学报,2009,14(2):27-31.
[19]Wang DP,Meng H,Guan XL,Luo XG.Identification of bacillus amyloliquefaciens BI2 and its antagonistic activity against Aspergillus flavus.Journal of Tianjin University of Science&Technology,2010,25(6):5-9.(in Chinese)王德培,孟慧,管叙龙,罗学刚.解淀粉芽孢杆菌BI2的鉴定及其对黄曲霉的抑制作用.天津科技大学学报,2010,25(6):5-9.
[20]Rao SQ,Chen SY,Gao L,Yin YQ,Yang ZQ,Fang WM.Screening,identification,and antifungal properties of a bacterial strain F1 with antagonistic activities against Aspergillus flavus.Modern Food Science and Technology,2015,31(12):99-105.(in Chinese)饶胜其,陈素雅,高璐,尹永祺,杨振泉,方维明.一株黄曲霉拮抗细菌F1的筛选、鉴定及其抑菌特性.现代食品科技,2015,31(12):99-105.
[21]Li CB,Li WM,Yang WH,Li HX,Liu XH,Cao YS.Isolation and characterisation of an aflatoxin B1-degrading bacterium.Acta Microbiologica Sinica,2012,52(9):1129-1136.(in Chinese)李超波,李文明,杨文华,李海星,刘晓华,曹郁生.黄曲霉毒素B1降解菌的分离鉴定及其降解特性.微生物学报,2012,52(9):1129-1136.
[22]韩愈杰.黄曲霉拮抗菌株的分离筛选及对黄曲霉毒素的降解效果.河北农业大学硕士学位论文,2015.
[23]Yue XY,Li ZG,Hao XZ,Xu J,Liu XD,Niu TG.Simulation of effect of main ecological factors on radial growth of Aspergillus flavus during storage period of corn.Transactions of the Chinese Society of Agricultural Engineering,2013,29(18):269-276.(in Chinese)岳晓禹,李自刚,郝修振,徐军,刘相东,牛天贵.主要生态因子对贮藏玉米中黄曲霉生长影响的模拟.农业工程学报,2013,29(18):269-276.
[24]Zhang P,He PF,Li ZX.Isolation and characterisation of aflatoxigen strains in corn.Southwest China Journal of Agricultural Sciences,2017,(30):146-150.(in Chinese)张佩,何鹏飞,李智新.玉米中产黄曲霉毒素菌株的分离与鉴定.西南农业学报(增刊),2017,(30):146-150.
[25]Shi JY,Ning H,Xu R,Gao ZX,Yu Y,Liu B,Ke YP,Sun Q.Inhibitory effect on the growth and aflatoxin-production of Aspergillus flavus by ClO2 and its application in corn of high water content.China Measurement&Test,2017,43(10):47-52.(in Chinese)史健阳,宁华,徐冉,高珍璇,余洋,刘保,柯永培,孙群.二氧化氯抑制黄曲霉菌生长和产毒能力及其在高水分玉米中的应用.中国测试,2017,43(10):47-52.