降解DON毒素的绿脓杆菌的分离、鉴定及其在玉米脱毒中的应用
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摘要
脱氧雪腐镰刀菌烯醇(DON)是污染谷物及其制品的最常见的真菌毒素之一,严重威胁谷物食品的安全性和经济价值。利用微生物降解对粮食和饲料中真菌毒素进行脱毒处理的方法具有潜在的应用价值。利用禾谷镰刀菌发酵产生的DON粗提物为唯一碳源从土样中筛选到了一株可高效降解DON的微生物,经16S rDNA序列比对分析确定该菌株为绿脓杆菌。该菌株可在48 h内降解64.6%的DON;对DON的降解起作用的物质主要存在于发酵上清液中,蛋白酶K处理发酵上清液可降低其毒素降解效率,初步判断上清液中存在可降解DON的酶类。利用超声波辅助提取DON污染的玉米中毒素及毒素降解试验表明:经3次提取处理,玉米中的呕吐毒素含量可降低99.0%以上,将绿脓杆菌接种于毒素提取液中72 h后呕吐毒素浓度可降低至50.0%。绿脓杆菌在粮食脱毒处理中具有较大的应用潜力。
Deoxynivalenol(DON)is a mycotoxin commonly contaminating cereal grains and grain products,which seriously threaten the safety and economic value of cereal food. Detoxification treatment of mycotoxins in cereal grains and feedstock by microbial degradation is an alternative approach with great potential. In this study,a microbial strain with DON degradation capacity was screened using the medium containing crude DON as sole carbon source extracted from corn inoculated with Fusarium graminearum. The sequence alignment of 16 S r DNA of the microbial strain demonstrated that it is Pseudomonas aeruginosa. This strain could degrade 64.6%DON after incubation at 35 ℃ for 72 h. The DON-degrading activity of Pseudomonas aeruginosa was predominantly attributed to the cell-free supernatant and this activity was found to be heat stable but sensitive to proteinase K treatment,indicating that the extracellular proteins or enzymes were responsible for the DON degradation. The DON was extracted from corn inoculated with F. graminearum with ultrasonic assisted extraction and DON degradation by Pseudomonas aeruginosa was tested,and it was found that the DON in corn reduced above 99.0% after extracted three times and 50.0% DON in corn extract was degraded by Pseudomonas aeruginosa after incubation at 35 ℃ for 72 h. This study provides an experimental basis for the detoxification treatment of DON contaminated cereal grain and feedstock by Pseudomonas aeruginosa.
Deoxynivalenol(DON)is a mycotoxin commonly contaminating cereal grains and grain products,which seriously threaten the safety and economic value of cereal food. Detoxification treatment of mycotoxins in cereal grains and feedstock by microbial degradation is an alternative approach with great potential. In this study,a microbial strain with DON degradation capacity was screened using the medium containing crude DON as sole carbon source extracted from corn inoculated with Fusarium graminearum. The sequence alignment of 16 S r DNA of the microbial strain demonstrated that it is Pseudomonas aeruginosa. This strain could degrade 64.6%DON after incubation at 35 ℃ for 72 h. The DON-degrading activity of Pseudomonas aeruginosa was predominantly attributed to the cell-free supernatant and this activity was found to be heat stable but sensitive to proteinase K treatment,indicating that the extracellular proteins or enzymes were responsible for the DON degradation. The DON was extracted from corn inoculated with F. graminearum with ultrasonic assisted extraction and DON degradation by Pseudomonas aeruginosa was tested,and it was found that the DON in corn reduced above 99.0% after extracted three times and 50.0% DON in corn extract was degraded by Pseudomonas aeruginosa after incubation at 35 ℃ for 72 h. This study provides an experimental basis for the detoxification treatment of DON contaminated cereal grain and feedstock by Pseudomonas aeruginosa.
引文
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[13] ALTALHI A D,EL-DEEB B. Localization of zearalenone detoxi覲cation gene(s)in p ZEA-1plasmid of P. putida ZEA-1 and expressed in Escherichia coli[J]. Journal of Hazardous Materials,2009,161:1166-1172.
[14] ALEXANDER N J.The Tri101 story:engineering wheat and barley to resist Fusarium head blight[J]. World Mycotoxin Journal,2008,1:31-37.
[2]尚艳娥.真菌毒素检测技术研究进展[J].北京工商大学学报(自然科学版),2012,11(6):2-3.
[3] GOSWANMI R S,XU J R,TRAIL F,et al.Genomic analysis of hostpathogen interaction between Fusarium graminearum and wheat during early stage of disease development[J].Microbiology,2006,152(6):1877-1890.
[4] GB 2761—2017,食品中真菌毒素限量[S].
[5]樊平声,沙国栋,沈培银,等.脱氧雪腐镰刀菌烯醇毒性和检测方法研究进展[J].检验检疫学刊,2010(1):2-4.
[6] ROTTER B A. Toxicology of dexoynivalenol(vomitoxin)[J]. Journal of Toxicology Enviromental Health Part A,1996,48(1):1-34.
[7]余祖华,丁轲,刘赛宝,等.一株降解呕吐毒素蜡样芽孢杆菌的筛选与鉴定[J].食品科学,2016(2):3-4.
[8] MICHIHIRO ITO,IKUO SATO,MOTOO KOITABASHI,et al.A novel actinomycete derived from wheat heads degrades deoxynivalenol in the grain of wheat and barley affected by Fusarium head blight[J]. Environmental Biotechnology,2012,10:2-6.
[9] HATHOUT A S,ALY S E. Biological detoxification of mycotoxins:A review[J].Annals of Microbiology,2014,64:905-919.
[10] GUAN S,JI C,ZHOU T,et al. A覲atoxin B1degradation by Stenotrophomonas Maltophilia and other microbes selected using coumarinmedium[J].International Journal of Molecular Sciences,2008,9:1489-1503.
[11]吴娱,王开萍,唐正江,等.米曲霉Aspergillus oryzae As-W.6对脱氧雪腐镰刀菌烯醇的降解效果[J].食品科学,2016,37:2-4.
[12] KIMURA M,KANEKO I,KOMIYAMA M,et al.Trichothecene 3-O-acetyltransferase protects both the producing organism and transformed yeast from related mycotoxins[J].Journal of Biological Chemistry,2007,273:1654-1661.
[13] ALTALHI A D,EL-DEEB B. Localization of zearalenone detoxi覲cation gene(s)in p ZEA-1plasmid of P. putida ZEA-1 and expressed in Escherichia coli[J]. Journal of Hazardous Materials,2009,161:1166-1172.
[14] ALEXANDER N J.The Tri101 story:engineering wheat and barley to resist Fusarium head blight[J]. World Mycotoxin Journal,2008,1:31-37.