玉米赤霉烯酮降解菌的分离、鉴定及其适宜降解条件的研究

doi:10.16431/j.cnki.1671-7236.2020.09.013

摘要/Abstract

摘要： 试验旨在利用微生物降解法对饲料中存在的玉米赤霉烯酮（zearalenone，ZEN）进行快速、有效的消减。从不同地区采集了牛粪、羊粪及土壤样品共70份，并从中分离出164株菌株。利用环戊酮作为唯一碳源对这164株菌株进行初筛，再以ZEN作为唯一碳源对初筛菌株进行复筛并获得了8株ZEN降解菌株。采用高效液相色谱法（HPLC）对复筛得到的8株ZEN降解菌株进行ZEN降解率的测定，得到1株降解效果良好的菌株即NF-PJ-5号菌株。该菌株经16S rDNA序列比对、构建系统发育树、菌落形态观察及生理生化试验鉴定后确定为多食鞘氨醇杆菌（Sphingobacterium multivorum）。分别研究该菌株在不同温度、不同pH及不同菌液浓度条件下对ZEN的降解能力，评估菌株性质；同时，初步探究该菌株对ZEN的清除机理。结果显示，在28~32 ℃，pH 6.5~7.5，菌液浓度在2×10⁹ CFU/mL以上时，该菌株对ZEN保持较高的降解率。在30 ℃、pH 7.0、160 r/min培养48 h后，该菌株对初始浓度为10 μg/mL的ZEN降解率达到83%。综上，本试验筛选到1株ZEN降解能力较强的菌株，经鉴定为多食鞘氨醇杆菌。初步推测该菌株可通过产生胞外酶降解ZEN，同时该菌细胞壁对ZEN具有一定吸附能力。

关键词: 玉米赤霉烯酮; 微生物降解; 多食鞘氨醇杆菌; 鉴定

Abstract: The aim of this study was to reduce zearalenone (ZEN) in feed by bio-degradation rapidly and effectively.A total of 70 samples of cow manure,sheep manure and soil were collected from different areas and then 164 strains were isolated from these samples.Cyclopentanone was used as the only carbon source to preliminarily screen the 164 strains isolated from the collected samples,and the second-screening was performed using zearalenone as the only carbon source.As a result,8 ZEN-degrading strains were obtained.The ZEN-degrading efficiency of 8 strains was determined by high performance liquid chromatography (HPLC),and a strain with high ZEN-degrading,namely NF-PJ-5 strain.The strain was identified as Sphingobacterium multivorum by 16S rDNA sequence comparison,phylogenetic tree construction,colony morphology observation and physiological and biochemical identification.The ability of NF-PJ-5 strain to degrade zearalenone at different temperature,different pH and different concentration of bacterial solution were studied.In addition,the mechanism of ZEN degradation by this strain was studied.The results showed that at 28-32 ℃,pH 6.5-7.5,and the concentration of bacterial solution above 2×10⁹ CFU/mL,the strain could maintain a high degradation rate.Besides,the strain could degrade 83% ZEN with an initial concentration of 10 μg/mL,after 48 h culture at 30 ℃,pH 7.0,and a shaking speed of 160 r/min.From the above,a highly efficient ZEN-degrading strain was screened in this study,and it was identified as Sphingobacterium multivorum.It was preliminarily speculated that the strain could degrade ZEN by producing extracellular enzyme,and the cell wall of the strain had a certain adsorption capacity for ZEN.

Key words: zearalenone; bio-degradation; Sphingobacterium multivorum; identification

中图分类号:

S816.3

金博文, 徐长春, 李根, 李晓宇, 王丽丽, 李纪彬, 徐永平. 玉米赤霉烯酮降解菌的分离、鉴定及其适宜降解条件的研究[J]. 中国畜牧兽医, 2020, 47(9): 2808-2815.

JIN Bowen, XU Changchun, LI Gen, LI Xiaoyu, WANG Lili, LI Jibin, XU Yongping. Isolation,Identification and Its Suitable Degradation Conditions of a Zearalenone-degrading Bacteria[J]. China Animal Husbandry and Veterinary Medicine, 2020, 47(9): 2808-2815.

参考文献

[1] STOB M,BALDWIN R S,TUITE J,et al.Isolation of an anabolic,uterotrophic compound from corn infected with Gibberella zeae[J].Nature,1962,196(4861):1318.
[2] PÉTER H,ISTVÁN S,MÁTYÁS C,et al.Biodegra-dation of aflatoxin-B₁ and zearalenone by Streptomyces sp.collection[J].International Biodeterioration & Biodegradation,2016,108:48-56.
[3] URRY W H,WEHRMEISTER H L,HODGE E B,et al.The structure of zearalenone[J].Tetrahedron Letters,1966,7(27):3109-3114.
[4] PLACINTA C M,D″MELLO J P F,MACDO-NALD A M C.A review of worldwide contamination of cerealgrains and animal feed with Fusarium mycotoxins[J].Animal Feed Science and Technology,1999,78(1-2):21-37.
[5] WANG Y,ZHANG J,WANG Y L,et al.Isolation and characterization of the Bacillus cereus BC7 strain,which is capable of zearalenone removal and intestinal flora modulation in mice[J].Toxicon:Official Journal of the International Society on Toxinology,2018,155:9-20.
[6] DESJARDINS A.Mycotoxins:Risks in plant,animal,and human systems[J].AAHE-ERIC/Higher Education Research Report,2003,9(7):48-50.
[7] 张立阳,刘帅,赵雪娇,等.不同吸附材料及改性处理对三种常见霉菌毒素脱毒效果研究[J].东北农业大学学报,2018,49(9):20-28. ZHANG L Y,LIU S,ZHAO X J,et al.Study on detoxification effect of different adsorption materials and modification treatments to three mycotoxins[J].Journal of Northeast Agricultural University,2018,49(9):20-28.(in Chinese)
[8] SCHATZMAYR G,STREIT E.Global occurrence of mycotoxins in the food and feed chain:Facts and figures[J].World Mycotoxin Journal,2013,6(3):213-222.
[9] OBREMSKI K,GAJECKI M,ZWIERZCHOWSKI W,et al.Influence of zearalenone on reproductive system cell proliferation in gilts[J].Polish Journal of Veteri-nary Sciences,2003,6(4):239-245.
[10] 梁梓森,马勇江,刘长永,等.玉米赤霉烯酮对小鼠肝脏及肾脏的毒性作用[J].中国兽医学报,2010,30(5):673-676. LIANG Z S,MA Y J,LIU C Y,et al.In vivo toxicity of zearalenone on liver and kidney in mice[J].Chinese Journal of Veterinary Science,2010,30(5):673-676.(in Chinese)
[11] 姜淑贞,孙华,黄丽波,等.不同水平玉米赤霉烯酮对断奶仔猪血清代谢产物和肝肾组织病理学影响[J].中国农业科学,2014,47(18):3708-3715. JIANG S Z,SUN H,HUANG L B,et al.Effects of zearalenone contaminated diets on serum metabolite and histopathology of liver and kidney in weaned piglets[J].Scientia Agricultura Sinica,2014,47(18):3708-3715.(in Chinese)
[12] ZHENG W,HUANG Q,PAN S,et al.Regulation of oncogenes and gap junction intercellular communi-cation during the proliferative response of zearalenone in TM3 cells[J].Human & Experimental Toxicology,2017,36(7):701-708.
[13] 时从来,李金贵.玉米赤霉烯酮毒性及其脱毒方法研究进展[J].现代牧业,2018,2(3):35-38. SHI C L,LI J G.Research progress on toxicity and detoxification of zearalenone[J].Modern Animal Husbandry,2018,2(3):35-38.(in Chinese)
[14] 徐子伟,万晶.饲料霉菌毒素吸附剂研究进展[J].动物营养学报,2019,31(12):5391-5398. XU Z W,WAN J.Advances in research on adsorbents of mycotoxins in feed[J].Chinese Journal of Animal Nutrition,2019,31(12):5391-5398.(in Chinese)
[15] 计成.饲料中玉米赤霉烯酮的生物降解[J].动物营养学报,2014,26(10):2949-2955. JI C.Biodegradation of zearalenone in feeds[J].Chinese Journal of Animal Nutrition,2014,26(10):2949-2955.(in Chinese)
[16] MOLNAR O,SCHATZMAYR G,FUCHS E,et al.Trichosporon mycotoxinivorans sp.nov.,a new yeast species useful in biological detoxification of various mycotoxins[J].Systematic and Applied Micro-biology,2004,27(6):661-671.
[17] CHO K J,KANG J S,CHO W T,et al.In vitro degradation of zearalenone by Bacillus subtilis[J].Biotechnology Letters,2010,32(12):1921-1924.
[18] TAN H,ZHANG Z M,HU Y C,et al.Isolation and characterization of Pseudomonas otitidis TH-N1 capable of degrading zearalenone[J].Food Control,2015,47:285-290.
[19] SUN X L,HE X X,XUE K S,et al.Biological detoxification of zearalenone by Aspergillus niger strain FS10[J].Food and Chemical Toxicology 2014,72:76-82.
[20] LUIZ K,ABRUNHOSA L,KELLY K,et al.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.
[21] 徐天宇,胡苏莹,周峻岗,等.微杆菌Microbacterium sp.FY1538降解赤霉烯酮的活性研究[J].复旦学报(自然科学版),2016,55(2):223-231. XU T Y,HU S Y,ZHOU J G,et al.Analysis of zearalenone-degrading activities of Microbacterium sp.FY1538[J].Journal of Fudan University (Natural Science),2016,55(2):223-231.(in Chinese)
[22] 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 Microbiology & Biotechnology,2011,27(5):1035-1043.
[23] TINYIRO S E,YAO W,SUN X,et al.Scavenging of zearalenone by Bacillus strains in vitro[J].Research Journal of Microbiology,2011,6(3):304-309.
[24] CSERHÁTI M,KRISZT B,KRIFATON C,et al.Mycotoxin degradation profile of Rhodococcus strains[J].International Journal of Food Microbiology,2013,166(1):176-185.
[25] TAN H,HU Y C,HE J,et al.Zearalenone degradation by two Pseudomonas strains from soil[J].Mycotoxin Research,2014,30(4):191-196.
[26] 翟晓红,陈振明.环己酮单加氧酶的克隆表达及酶学性质分析[J].杭州师范大学学报(自然科学版),2012,5:17-22. ZHAI X H,CHEN Z M.Cloning expression and enzyme characterization of a cyclohexanone monooxy-genase[J].Journal of Hangzhou Normal University(Natural Science Edition),2012,5:17-22.(in Chinese)
[27] 苏志俊,刘永涛,徐春娟,等.一株溴氰菊酯降解菌的分离鉴定及降解特性研究[J].淡水渔业,2019,49(1):100-108. SU Z J,LIU Y T,XU C J,et al.Isolation,identification and degradation characteristics of a deltamethrin-degrading bacterial strain[J].Freshwater Fisheries,2019,49(1):100-108.(in Chinese)
[28] 陆源源.多食鞘氨醇菌活性污泥法降解水中甾体雌激素机理研究[D].南京:南京理工大学,2012. LU Y Y.Study on degradatation of steroid estrogens by activated sludge and a Sphingo.multivorum bacterium[D].Nanjing:Nanjing University of Science & Technology,2012.(in Chinese)
[29] ELNEZAMI H,POLYCHRONAKI N,LEE Y,et al.Chemical moieties and interactions involved in the binding of zearalenone to the surface of Lactobacillus rhamnosus strains GG[J].Journal of Agricultural and Food Chemistry,2004,52(14):4577-4581.