阿维菌素降解菌的筛选及其降解特性研究
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摘要
阿维菌素被视为目前最为优良、应用最广泛的兽用驱虫药,大量的应用已导致该药在环境中的残留,其残留不仅会诱导耐药菌株的形成,还会破坏生态环境,因此必将出现严重的污染问题。微生物降解是近些年来发展起来的处理环境中残留药物的有效方法之一。本文以研究阿维菌素降解菌的筛选为主线,建立了高效降解菌的筛选模型并进行了菌株降解特性的研究。
本文首先建立了无机盐培养基中阿维菌素含量的HPLC检测方法。采用乙腈提取阿维菌素,以甲醇:水(92:8,v/v)为流动相进行HPLC分析。阿维菌素在培养基中的回收率分别为82.33%-94.9%。日内变异系数范围为2.3-3.0%,日内变异系数范围为3.0-5.0%,LOQ为1mg/L。
以相对贫乏的培养基作为限制性条件构建菌株筛选模型。采用直接涂布分离法得到4株菌,其中AVM1菌株降解率相对较高,经革兰氏染色观察和生理生化试验,初步鉴定该菌株为蜡样芽孢杆菌。
AVM1菌株在pH7.0,温度为35℃,底物浓度40mg/L,装样量为100mL时,能够维持较高的降解率。添加少量的外加碳、氮源能够提高菌株对阿维菌素的降解率。在添加牛肉膏的条件下,AVM1菌株对初始浓度为40mgL/的阿维菌素6d的降解率为41%;金属离子对菌株降解阿维菌素的速率也有很大的影响,本实验研究了7种金属离子对菌株降解阿维菌素的影响,其中添加0.1%的Cu2+时可提高菌株对阿维菌素的降解率,AVM1菌株对初始浓度为40mgL/的阿维菌素6d的降解率为43.5%。
本实验还初步研究了AVM1菌株对阿维菌素类另外一种药物伊维菌素降解情况,试验结果表明,该菌株对伊维菌素没有降解能力。
Abamectin has been considered as the best and widely used veterinary anthelmintic at present, drug residues to environment have been brought by a devil of applications, not only formation of resistant strains will be induced, but also ecological environment will be destroyed by these residues, without doubt, serious pollution problems will be followed. The technology of biodegradation has been developed rapidly in recent years, and it has become one of the most effective ways to solve problem of drug residues in the environment. Screening model of the highly effective degeneration bacterium was established in the study, and degeneration characteristics of the strain were also studied.
A high performance liquid chromatography (HPLC) method was developed for determination of Avermectin (AVM).Ehylacetate was used for extracting Avermectin from medium. Mixture of methylate and H2O (92:8, V/V) was used as mobile phase in the HPLC analysis. The recoveries of Avermectin ranged from 86.95%~91.68% in medium, the coefficients of variation of AVM ranged from 2.3-3.0% and 3.0-5.0%, and the limit of quantitation (LOQ) of AVM was 0.5mg/L.
A highly effctive screening model was built,in which enrichment was condueted by adding a definite Avermectin into the innutriment medium. By using direct spreading method,four strains of bacteria were isolated from soil, among which one strain showed the Avermectin degradation ability and that was named as AVM1 strain. The strain was identified as Bacillus cereus.
The AVM1strain was considered as the goal strain for studying the generation characteristics. The AVM1 strain under the condition of pH7.0, temperature 35℃, the substrate concentration was the 40mg/L, and volume of medium was 100mL, a higher degeneration speed could be maintained. Adding carbon and nitrogen into the medium, degradation ability of the strain was enhanced. By adding soluble starch into the medium, the degeneration rate of the AVM1strain could be reached to 41% in the 40mg/L Avermectin solution for 6 days. The metal ions have a significant effect on Avermectin degeneration speed of the strain. In the study, the influence of 7 ions were investigated. The result showed that degradation ability of the strain was enhanced by adding 0.1% Cu2+, degeneration rate were 43.5%.
Research of degeneration of AVM1 strain to the Ivermectin was also made under the same situation in the study. The result indicated that the strain didn’t show degradation ability to the Ivermectin.
本文首先建立了无机盐培养基中阿维菌素含量的HPLC检测方法。采用乙腈提取阿维菌素,以甲醇:水(92:8,v/v)为流动相进行HPLC分析。阿维菌素在培养基中的回收率分别为82.33%-94.9%。日内变异系数范围为2.3-3.0%,日内变异系数范围为3.0-5.0%,LOQ为1mg/L。
以相对贫乏的培养基作为限制性条件构建菌株筛选模型。采用直接涂布分离法得到4株菌,其中AVM1菌株降解率相对较高,经革兰氏染色观察和生理生化试验,初步鉴定该菌株为蜡样芽孢杆菌。
AVM1菌株在pH7.0,温度为35℃,底物浓度40mg/L,装样量为100mL时,能够维持较高的降解率。添加少量的外加碳、氮源能够提高菌株对阿维菌素的降解率。在添加牛肉膏的条件下,AVM1菌株对初始浓度为40mgL/的阿维菌素6d的降解率为41%;金属离子对菌株降解阿维菌素的速率也有很大的影响,本实验研究了7种金属离子对菌株降解阿维菌素的影响,其中添加0.1%的Cu2+时可提高菌株对阿维菌素的降解率,AVM1菌株对初始浓度为40mgL/的阿维菌素6d的降解率为43.5%。
本实验还初步研究了AVM1菌株对阿维菌素类另外一种药物伊维菌素降解情况,试验结果表明,该菌株对伊维菌素没有降解能力。
Abamectin has been considered as the best and widely used veterinary anthelmintic at present, drug residues to environment have been brought by a devil of applications, not only formation of resistant strains will be induced, but also ecological environment will be destroyed by these residues, without doubt, serious pollution problems will be followed. The technology of biodegradation has been developed rapidly in recent years, and it has become one of the most effective ways to solve problem of drug residues in the environment. Screening model of the highly effective degeneration bacterium was established in the study, and degeneration characteristics of the strain were also studied.
A high performance liquid chromatography (HPLC) method was developed for determination of Avermectin (AVM).Ehylacetate was used for extracting Avermectin from medium. Mixture of methylate and H2O (92:8, V/V) was used as mobile phase in the HPLC analysis. The recoveries of Avermectin ranged from 86.95%~91.68% in medium, the coefficients of variation of AVM ranged from 2.3-3.0% and 3.0-5.0%, and the limit of quantitation (LOQ) of AVM was 0.5mg/L.
A highly effctive screening model was built,in which enrichment was condueted by adding a definite Avermectin into the innutriment medium. By using direct spreading method,four strains of bacteria were isolated from soil, among which one strain showed the Avermectin degradation ability and that was named as AVM1 strain. The strain was identified as Bacillus cereus.
The AVM1strain was considered as the goal strain for studying the generation characteristics. The AVM1 strain under the condition of pH7.0, temperature 35℃, the substrate concentration was the 40mg/L, and volume of medium was 100mL, a higher degeneration speed could be maintained. Adding carbon and nitrogen into the medium, degradation ability of the strain was enhanced. By adding soluble starch into the medium, the degeneration rate of the AVM1strain could be reached to 41% in the 40mg/L Avermectin solution for 6 days. The metal ions have a significant effect on Avermectin degeneration speed of the strain. In the study, the influence of 7 ions were investigated. The result showed that degradation ability of the strain was enhanced by adding 0.1% Cu2+, degeneration rate were 43.5%.
Research of degeneration of AVM1 strain to the Ivermectin was also made under the same situation in the study. The result indicated that the strain didn’t show degradation ability to the Ivermectin.
引文
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[3] Bruce JR,PaulKSL,MichaelM.Emerging chemicals of concern,Pharmaceuticals and personal care products(PPCPs) in Asia,with particular reference to Southern China[J].Marine Pollution Bulletin,2005,50:913-920.
[4]王向荣,方热军.饲用抗生素的应用现状、存在问题及其对策.湖南饲料.2006,6:19-27.
[5] ElmundG K,Morrison S M,Grant DW,et al. Role of excreted chlortetracycline in modifying the decomposition process in feedlot waste[J].Bulletin of Environmental Contamination and Toxicology,1971,6(2):129-135.
[6] Feinman SE,Matheson JCD raft environmental impact statement:Subtherapeutic antibacterial agents in animal feeds[M].Washington,DC:Food and Drug Administration,1978.372.
[7] Alcock R E,Sweetman A,Jones KC. Assessment of organic contaminant fate in waste water treatment plants.I:Selected compounds and physicochemical propertie [J].Chemosphere,1999,8(10):2247-2262.
[8] Beconi-Barker M G,Hornish R E,Vidmar T J,et al. Ceftiofur hydrochloride:plasma and tissue distribution in swine following intramuscular administration at various doses[J].Journal of Veterinary Pharmacology&Therapeutics,1996,19(3):192-199.
[9] Halling-ScrensenB,NielsenSN,LanzkyPF,IngerslevF,Lutzhcft HCH,Jcrgensen SE Occurence, fate and effects of pharmaceutical substances in the environment-A review.Chemosphere,1998,36(2):357-393.
[10] Gavalchin J,Katz SE.The persistence of face al-borne antibiotics in soil.Journal of AOAC International,1994,77(2):481-485.
[11] ElmundGK,MorrisonSM,GrantDW,Nevins MP. Role of excreted chlortetracycline in modifying the decomposition process in feedlot waste. Bull. Environ. Contam.Toxicol.,1971,6(2): 29 -135.
[12] Feinman SE,Matheson JC. Draft environmental impact statement:Subtherapeutic antibacterial agents in animal feeds.Washington,D.C.:Food and Drug Administration Department of Health,Education and Welfare Report,Food and Drug Administration,1978,372.
[13] Addison J B.Antibiotics in sediments and run-off water from feed lots.Residu Reviews,1984,92:1-28.
[14] Egerton J.E. Avermectins, a new family of potent anthelmintic agents: efficacy of the B,acomponent,Antimicrob. Agents Chemother.1979,15:372-378.
[15] Miller T.W, Chalet L.,Cole D.J., et al. Avermectins, a new family of anthelmintic agent: producing organism and fermentation, Antimicrob.Agents Chemother. 1979, 15:368-371.
[16]刘占民,马清河,刘荣欣.阿维菌素及其临床应用现状.中兽医医药杂志,1999, 18 (6): 17-19.
[17] Mckellar Q.A., Benchaoui H.A. Avermectins and milbermycins, Journal of Veterinary Pharmacologyand Therapeutics. 1996,19:331-351.
[18]沈寅初,杨慧心.杀虫抗生素Avermectin的开发及特性.农药译丛,1994, 16 (3) :1-13.
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