一株黄曲霉拮抗菌的筛选、鉴定及特性研究
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摘要
为获得拮抗黄曲霉和降解黄曲霉毒素的生物防治菌株,以烟台黄曲霉污染区域土壤为材料,采用稀释分离法和琼脂扩散法,筛选到一株对黄曲霉有抑制作用且可降解黄曲霉毒素的菌株Y-17-3。根据菌株的形态学特征、生理生化试验结合16S rDNA基因序列分析和系统发育树构建,确定菌株Y-17-3为空气芽孢杆菌(Bacillus aerius)。该菌株发酵液经硫酸铵沉淀的抗菌活性及稳定性试验,发现70%饱和硫酸铵沉淀的蛋白粗提物抑制黄曲霉活性最强,可降低黄曲霉孢子的萌发率,抑制菌丝的生长和菌丝球的形成,且该蛋白粗提物对温度、pH值和蛋白酶敏感。菌株Y-17-3能够降解黄曲霉毒素B_1,培养6 d时的降解率达90%。综上,所分离的空气芽孢杆菌Y-17-3菌株能够通过产生拮抗蛋白来抑制黄曲霉生长,且具有较强的黄曲霉毒素降解能力,在农业生物防治领域具有一定的应用前景。
To obtain bio-control bacteria that can effectively suppress the growth of Aspergillus flavus and degrade the aflatoxin, the antagonistic strain Y-17-3 was screened by the agar diffusion method from the soil contaminated with A. flavus in Yantai. The strain Y-17-3 was identified to be Bacillus aerius based on the morphological characteristics, physiological and biochemical experiments, and the 16 S rDNA gene sequence analysis followed by constructing phylogenetic tree. The crude protein extract precipitated at 70% saturated ammonium sulfate from the culture supernatant of strain Y-17-3 showed higher activity against A. flavus 3.2890. The spore germination rate of A. flavus 3.2890 treated with protein precipite was greatly reduced This active ingredient could inhibit the growth of mycelium and the formation of mycelium ball. The crude protein extract was sensitive to temperature, pH and protease. It could degrade aflatoxin B_1 by 90% after incubation at 37℃ for 6 d. The results showed that the strain B. aerius Y-17-3 isolated in this study can produce antagonistic proteins which inhibit the growth of A. flavus and was capable to degrade aflatoxins B_1 degradation. Therefore, this strain has the potential to be applied in the field of agricultural biological control.
To obtain bio-control bacteria that can effectively suppress the growth of Aspergillus flavus and degrade the aflatoxin, the antagonistic strain Y-17-3 was screened by the agar diffusion method from the soil contaminated with A. flavus in Yantai. The strain Y-17-3 was identified to be Bacillus aerius based on the morphological characteristics, physiological and biochemical experiments, and the 16 S rDNA gene sequence analysis followed by constructing phylogenetic tree. The crude protein extract precipitated at 70% saturated ammonium sulfate from the culture supernatant of strain Y-17-3 showed higher activity against A. flavus 3.2890. The spore germination rate of A. flavus 3.2890 treated with protein precipite was greatly reduced This active ingredient could inhibit the growth of mycelium and the formation of mycelium ball. The crude protein extract was sensitive to temperature, pH and protease. It could degrade aflatoxin B_1 by 90% after incubation at 37℃ for 6 d. The results showed that the strain B. aerius Y-17-3 isolated in this study can produce antagonistic proteins which inhibit the growth of A. flavus and was capable to degrade aflatoxins B_1 degradation. Therefore, this strain has the potential to be applied in the field of agricultural biological control.
引文
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[13] Afsah Hejri L. Saprophytic yeasts: Effective biocontrol agents against Aspergillus flavus[J]. International Food Research Journal, 2013, 20(6): 3403-3409
[14] Raksha Rao K, Vipin A Ⅴ, Hariprasad P, Anu Appaiah K A, Venkateswaran G. Biological detoxification of Aflatoxin B1 by Bacillus licheniformis CFR1 [J]. Food Control, 2017, 71(1): 234-241
[15] Pereyra M L G, Martínez M P, Petroselli G, Erra Balsells R, Cavaglieri L R. Antifungal and aflatoxin-reducing activity of extracellular compounds produced by soil Bacillus strains with potential application in agriculture [J]. Food Control, 2018, 85(3): 392-399
[16] Munimbazi C, Bullerman L B. Inhibition of Aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus [J]. Mycopathologia, 1997, 140(3): 163-169
[17] Akocak P B, Churey J J, Worobo R W. Antagonistic effect of chitinolytic Pseudomonas and Bacillus on growth of fungal hyphae and spores of aflatoxigenic Aspergillus flavus[J]. Food Bioscience, 2015, 10(1): 48-58
[18] Farzaneh M, Shi Z Q, Ghassempour A, Sedaghat N, Ahmadzadeh M, Mirabolfathy M, Javan-Nikkhah M. Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran [J]. Food Control, 2012, 23(1): 100-106
[19] 孙晓霞, 胡连霞, 姜彦芬, 陈志敏, 付琦. 降解黄曲霉毒素M1菌株筛选方法的建立与菌株鉴定[J]. 食品工业科技, 2017, 38(20):120-124
[20] 东秀珠, 蔡妙英. 常见细菌鉴定手册[M]. 北京: 科学出版社, 2001: 62-63
[21] R E 布坎南, N E 吉本斯. 伯杰鉴定手册(第八版) [M]. 中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组, 译. 北京: 科学出版社, 1995: 591-611
[22] Li J F, Li H F, Zhang Y Y, Duan X H, Liu J. Characterization of a bacteriocin-like substance produced from a novel isolated strain of Bacillus subtilis SLYY-3 [J]. Journal of Ocean University of China, 2014, 13(4): 995-999
[23] 姚彦坡, 张立田, 郑百芹, 张建民, 董李学, 项爱丽, 李爱军. 花生黄曲霉毒素污染生防细菌筛选及菌株B85-1的鉴定和抗菌活性[J]. 花生学报, 2016, 45(3): 20-26
[24] 张雪. Bacillus aerius S-7菌株纤溶酶的分离纯化及纤溶活性分析[D]. 保定: 河北农业大学, 2012
[25] Saun N K, Mehta P, Gupta R. Purification and physicochemical properties of lipase from thermophilic Bacillus aerius [J]. Journal of Oleo Science, 2014, 63(12): 1261-1268
[26] Moyne A L, Shelby R, Cleveland T E, Tuzun S. Baeillomycin D: an iturin with antifungal activity against Aspergillus flavus [J]. Journal of Appfied Microbiology, 2001, 90(4): 622-629
[27] Osouli S, Afsharmanesh H, Osouli S, Afsharmanesh H. To evaluate the effects of secondary metabolites produced by Bacillus subtilis mutant M419 against Papilio demoleus L. and Aspergillus flavus [J]. Acta Ecologica Sinica, 2016, 36(6): 492-496
[28] Kong Q, Shan S, Liu Q, Wang X, Yu F. Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium [J]. International Journal of Food Microbiology, 2010, 139(1/2): 31-35
[29] Afsharmanesh H, Ahmadzadehb M, Javan-Nikkhahb M, Behboudib K. Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation[J]. Crop Protection, 2014, 60(2): 83-92
[30] 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[J]. 微生物学报, 2012, 52(9): 1129-1136
[31] 关心, 何剑斌, 董双, 臧健, 龙淼. 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
[32] Xia X H, Zhang Y, Li M Y, Garba B, Zhang Q, Wang Y, Zhang H Y, Li P W. Isolation and characterization of a Bacillus subtilis strain with aflatoxin B1 biodegradation capability [J]. Food Control, 2017, 75(5): 92-98
[2] 李金芝. 黄曲霉拮抗菌株的筛选、鉴定及其活性物质的初步研究[D]. 青岛: 青岛科技大学, 2013
[3] 饶胜其, 陈素雅, 魏永峰, 高璐, 尹永祺, 杨振泉, 方维明. 解淀粉芽孢杆菌F1对黄曲霉的拮抗条件优化及其抑霉特性研究[J]. 食品与发酵工业, 2015, 41(12): 6-12
[4] 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
[5] da Rocha M E B, Freire F D O, Maia F B F, Guedes M Ⅰ F, Rondina D. Mycotoxins and their effects on human and animal health [J]. Food Control, 2014, 36(1): 159-165
[6] Siahmoshteh F, Siciliano Ⅰ, Banani H, HamidiEsfahani Z, Razzaghi A, byaneh M, Gullino M L, Spadaro D. Efficacy of Bacillus subtilis and Bacillus amyloliquefaciens in the control of Aspergillus parasiticus growth and Aflatoxins production on pistachio [J]. International Journal of Food Microbiology, 2017, 254(5): 47-53
[7] 王利敏, 邢福国, 吕聪, 刘阳. 复合植物精油防霉剂对玉米霉菌及真菌毒素的控制效果[J]. 核农学报, 2018, 32(4): 732-739
[8] Siciliano Ⅰ, Spadaro D, Prelle A, Vallauri D, Cavallero M, Garibaldi A, Gullino M. Use of cold atmospheric plasma to detoxify hazelnuts from aflatoxins [J]. Toxins, 2016, 8(5): 125-128
[9] Basappa S C, Shantha T. Methods for detoxification of Aflatoxins in foods and feeds-a critical appraisal[J]. Journal of Food Science and Technology, 1996, 33(2): 95-107
[10] Verheecke C, Liboz T, Mathieu F. Microbial degradation of Aflatoxin B1: Current status and future advances [J]. International Journal of Food Microbiology, 2016, 237(7): 1-9
[11] Samuel M S, Sivaramakrishna A, Mehta A. Degradation and detoxification of Aflatoxin B1 by Pseudomonas putida [J]. International Biodeterioration and Biodegradation, 2014, 86(1): 202-209
[12] Mishra H N, Das C. A review on biological control and metabolism of Aflatoxin [J]. Critical Reviews in Food Science and Nutrition, 2003, 43(3): 245-264
[13] Afsah Hejri L. Saprophytic yeasts: Effective biocontrol agents against Aspergillus flavus[J]. International Food Research Journal, 2013, 20(6): 3403-3409
[14] Raksha Rao K, Vipin A Ⅴ, Hariprasad P, Anu Appaiah K A, Venkateswaran G. Biological detoxification of Aflatoxin B1 by Bacillus licheniformis CFR1 [J]. Food Control, 2017, 71(1): 234-241
[15] Pereyra M L G, Martínez M P, Petroselli G, Erra Balsells R, Cavaglieri L R. Antifungal and aflatoxin-reducing activity of extracellular compounds produced by soil Bacillus strains with potential application in agriculture [J]. Food Control, 2018, 85(3): 392-399
[16] Munimbazi C, Bullerman L B. Inhibition of Aflatoxin production of Aspergillus parasiticus NRRL 2999 by Bacillus pumilus [J]. Mycopathologia, 1997, 140(3): 163-169
[17] Akocak P B, Churey J J, Worobo R W. Antagonistic effect of chitinolytic Pseudomonas and Bacillus on growth of fungal hyphae and spores of aflatoxigenic Aspergillus flavus[J]. Food Bioscience, 2015, 10(1): 48-58
[18] Farzaneh M, Shi Z Q, Ghassempour A, Sedaghat N, Ahmadzadeh M, Mirabolfathy M, Javan-Nikkhah M. Aflatoxin B1 degradation by Bacillus subtilis UTBSP1 isolated from pistachio nuts of Iran [J]. Food Control, 2012, 23(1): 100-106
[19] 孙晓霞, 胡连霞, 姜彦芬, 陈志敏, 付琦. 降解黄曲霉毒素M1菌株筛选方法的建立与菌株鉴定[J]. 食品工业科技, 2017, 38(20):120-124
[20] 东秀珠, 蔡妙英. 常见细菌鉴定手册[M]. 北京: 科学出版社, 2001: 62-63
[21] R E 布坎南, N E 吉本斯. 伯杰鉴定手册(第八版) [M]. 中国科学院微生物研究所《伯杰细菌鉴定手册》翻译组, 译. 北京: 科学出版社, 1995: 591-611
[22] Li J F, Li H F, Zhang Y Y, Duan X H, Liu J. Characterization of a bacteriocin-like substance produced from a novel isolated strain of Bacillus subtilis SLYY-3 [J]. Journal of Ocean University of China, 2014, 13(4): 995-999
[23] 姚彦坡, 张立田, 郑百芹, 张建民, 董李学, 项爱丽, 李爱军. 花生黄曲霉毒素污染生防细菌筛选及菌株B85-1的鉴定和抗菌活性[J]. 花生学报, 2016, 45(3): 20-26
[24] 张雪. Bacillus aerius S-7菌株纤溶酶的分离纯化及纤溶活性分析[D]. 保定: 河北农业大学, 2012
[25] Saun N K, Mehta P, Gupta R. Purification and physicochemical properties of lipase from thermophilic Bacillus aerius [J]. Journal of Oleo Science, 2014, 63(12): 1261-1268
[26] Moyne A L, Shelby R, Cleveland T E, Tuzun S. Baeillomycin D: an iturin with antifungal activity against Aspergillus flavus [J]. Journal of Appfied Microbiology, 2001, 90(4): 622-629
[27] Osouli S, Afsharmanesh H, Osouli S, Afsharmanesh H. To evaluate the effects of secondary metabolites produced by Bacillus subtilis mutant M419 against Papilio demoleus L. and Aspergillus flavus [J]. Acta Ecologica Sinica, 2016, 36(6): 492-496
[28] Kong Q, Shan S, Liu Q, Wang X, Yu F. Biocontrol of Aspergillus flavus on peanut kernels by use of a strain of marine Bacillus megaterium [J]. International Journal of Food Microbiology, 2010, 139(1/2): 31-35
[29] Afsharmanesh H, Ahmadzadehb M, Javan-Nikkhahb M, Behboudib K. Improvement in biocontrol activity of Bacillus subtilis UTB1 against Aspergillus flavus using gamma-irradiation[J]. Crop Protection, 2014, 60(2): 83-92
[30] 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[J]. 微生物学报, 2012, 52(9): 1129-1136
[31] 关心, 何剑斌, 董双, 臧健, 龙淼. 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
[32] Xia X H, Zhang Y, Li M Y, Garba B, Zhang Q, Wang Y, Zhang H Y, Li P W. Isolation and characterization of a Bacillus subtilis strain with aflatoxin B1 biodegradation capability [J]. Food Control, 2017, 75(5): 92-98