牧草中抗生素残留检测及来源的初步研究
摘要
抗生素（Antibiotics)一般被用作治疗疾病的药物和饲料添加剂，以治疗动物的疾病和增加饲料的利用率，所以抗生素的残留一般被公认为来源于动物饲料添加剂和治疗动物疾病的药物。但是在2009年对蒙古国草原上的110多种天然牧草(未作任何抗生素的添加)进行检测，结果发现：A.macrocephala、ArtemisiaSieversiana等20余种牧草中检测出氯霉素，氯霉素含量为0.08ug/kg—3.073ug/kg。动物实验表明：饲喂了这些牧草后，在动物组织中能检测出氯霉素，动物组织中氯霉素残留量为0.035ug/kg—4.12ug/kg。这项研究表明某些动物产品中的抗生素残留究其原因来源于植物体自身和寄生的微生物，这将影响到欧盟、美国等国家对于食物中抗生素残留限量的立法。本文主要是建立适合于各种草样基质的5种抗生素残留检测方法，筛选出可通过灌溉水方式迁移的抗生素种类和牧草品种，这种迁移方式下牧草中抗生素残留行为，同时探讨存在产抗生素的微生物条件下，对该残留行为的影响。主要研究内容及结果如下：
（1）建立牧草中土霉素、四环素、罗红霉素、氯霉素、青霉素五种抗生素的反相液相色谱-串联质谱（HPLC-MS/MS）检测方法。五种抗生素目标物在20μg/kg~500μg/kg的范围内线性关系良好，相关系数在0.9901~0.9998之间。在50μg/kg、100μg/kg、200μg/kg三个添加水平上，平均回收率在80%~110%之间，相对标准偏差在5.8%~15.0%之间，五种抗生素的检出限是0.013~0.18μg/kg，定量限是0.04~0.59μg/kg。
（2）移栽牧草和种植的牧草做灌溉水中抗生素添加的施入实验，进行ELASA法和LC/MS/MS方法检测。实验证明灌溉水中添加抗生素的施入方式能够使牧草中残留抗生素，不同的抗生素吸附不同的牧草，土霉素和四环素对黑麦草、苜蓿和茅草的吸附能力好；泰乐菌素主要能寄生于黑麦草、苜蓿和菊苣等牧草中；青霉素、氯霉素和红霉素对黑麦草、苜蓿和三叶草的吸附性好；链霉素对黑麦草、菊苣、三叶草和无芒雀麦的吸附能力好；卡那霉素对黑麦草、苜蓿和菊苣的吸附能力好；抗生素能的残留还与施入的抗生素浓度有关系，浓度越大残留量越多，如土霉素施入量为200mg时，黑麦草中土霉素的残留为24.2μg/kg,当施入量为100mg时残留量为13.2μg/kg；施入同一浓度的抗生素时，不同时期检测到的抗生素残留量也不同，如同时施入四环素100mg于灌溉水中，7天、15天、30天、45天分别检测到的抗生素残留浓度为19.2、16.5、10.3、2.1μg/kg，呈现一个递减的趋势，可见牧草对抗生素具有消解作用。
（3）菌体自身和发酵的培养基中均检测到作为其次级代谢产物的抗生素，将含有液体培养基的菌以灌溉方式施入到牧草土壤，检测到牧草中有抗生素的残留。
Antibiotics are used as the drug to treatment the animals'disease and as the feed additives,so the antibiotics reduces are recognized as coming from the drugs and the feed additives.In the environment, antibiotics will present in forage with different ways, or be produced by microorganism,which endangers Human Health.In2009,Approximately110herbs and grass were analysed and found that in A.macrocephala、Artemisia Sieversiana et al.26samples proved to contain CAP at levels ranging from0.08to3.073μg/kg.With Animal experiment found products of animal origin contain CAP at levels ranging from0.035to4.12ug/kg.The results of this study demonstrate that noncompliant findings in animal derived food products may in part be due to the natural occurrence of chloramphenicol in plant material.This has implications for the application of current EU and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.The main object in our study is to discuss how to happen about the antibiotics in herbs and grass,and establish a method to detect it,details are as follows:
(1) Develop LC/MS/MS method to determinate antibiotics in grass and herbs.The mean recoveries of all antibiotics were in range from80%to110%, relative standard deviation were from5.8%to15%at the20μg/kg,50μg/kg and100μg/kg Spike level.The limit of determination of antibiotics is between0.013μg/kg-0.18μg/kg,limit of quantity is between0.04μg/kg-0.59μg/kg.
(2) Transplanting of forage and planting grass do the experiment add antibiotics into the water,use ELISA and LC-MS/MS methods for detection.The experiment proves that the antibiotic residues in the grass.Different antibiotics adsorption on different forage.The oxytetracycline and tetracycline mainly adsorption on Lolium perenne,alfalfa and couch grass.The tylosin adsorption on Lolium perenne,alfalfa and chicory.The penicillin,chloramphenicol and erythromycin residue on Lolium perenne, alfalfa and clover.The streptomycin residues on Lolium perenne, clover, chicory and bromus inermis.The kanamycin mainly adsorption on Lolium perenne,alfalfa and chicory. The antibiotic residual has the relationship with the amount of adding to the water,and the more addition the higher residual.When add oxytetracyclin200mg to the water,the residual in grass about24.2μg/kg,and the residual is13.2μg/kg when add100mg.When add the same concentration antibiotic to the water,test the residual in forage on different periods and it shows an decline trend.The forage has digestion effect on antibiotics.
(3) The bacteria could produce antibitics itself, and its medium also can find antibiotics.The herbs are irrigated with the bacteria could be detected antibiotics residue.
（1）建立牧草中土霉素、四环素、罗红霉素、氯霉素、青霉素五种抗生素的反相液相色谱-串联质谱（HPLC-MS/MS）检测方法。五种抗生素目标物在20μg/kg~500μg/kg的范围内线性关系良好，相关系数在0.9901~0.9998之间。在50μg/kg、100μg/kg、200μg/kg三个添加水平上，平均回收率在80%~110%之间，相对标准偏差在5.8%~15.0%之间，五种抗生素的检出限是0.013~0.18μg/kg，定量限是0.04~0.59μg/kg。
（2）移栽牧草和种植的牧草做灌溉水中抗生素添加的施入实验，进行ELASA法和LC/MS/MS方法检测。实验证明灌溉水中添加抗生素的施入方式能够使牧草中残留抗生素，不同的抗生素吸附不同的牧草，土霉素和四环素对黑麦草、苜蓿和茅草的吸附能力好；泰乐菌素主要能寄生于黑麦草、苜蓿和菊苣等牧草中；青霉素、氯霉素和红霉素对黑麦草、苜蓿和三叶草的吸附性好；链霉素对黑麦草、菊苣、三叶草和无芒雀麦的吸附能力好；卡那霉素对黑麦草、苜蓿和菊苣的吸附能力好；抗生素能的残留还与施入的抗生素浓度有关系，浓度越大残留量越多，如土霉素施入量为200mg时，黑麦草中土霉素的残留为24.2μg/kg,当施入量为100mg时残留量为13.2μg/kg；施入同一浓度的抗生素时，不同时期检测到的抗生素残留量也不同，如同时施入四环素100mg于灌溉水中，7天、15天、30天、45天分别检测到的抗生素残留浓度为19.2、16.5、10.3、2.1μg/kg，呈现一个递减的趋势，可见牧草对抗生素具有消解作用。
（3）菌体自身和发酵的培养基中均检测到作为其次级代谢产物的抗生素，将含有液体培养基的菌以灌溉方式施入到牧草土壤，检测到牧草中有抗生素的残留。
Antibiotics are used as the drug to treatment the animals'disease and as the feed additives,so the antibiotics reduces are recognized as coming from the drugs and the feed additives.In the environment, antibiotics will present in forage with different ways, or be produced by microorganism,which endangers Human Health.In2009,Approximately110herbs and grass were analysed and found that in A.macrocephala、Artemisia Sieversiana et al.26samples proved to contain CAP at levels ranging from0.08to3.073μg/kg.With Animal experiment found products of animal origin contain CAP at levels ranging from0.035to4.12ug/kg.The results of this study demonstrate that noncompliant findings in animal derived food products may in part be due to the natural occurrence of chloramphenicol in plant material.This has implications for the application of current EU and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.The main object in our study is to discuss how to happen about the antibiotics in herbs and grass,and establish a method to detect it,details are as follows:
(1) Develop LC/MS/MS method to determinate antibiotics in grass and herbs.The mean recoveries of all antibiotics were in range from80%to110%, relative standard deviation were from5.8%to15%at the20μg/kg,50μg/kg and100μg/kg Spike level.The limit of determination of antibiotics is between0.013μg/kg-0.18μg/kg,limit of quantity is between0.04μg/kg-0.59μg/kg.
(2) Transplanting of forage and planting grass do the experiment add antibiotics into the water,use ELISA and LC-MS/MS methods for detection.The experiment proves that the antibiotic residues in the grass.Different antibiotics adsorption on different forage.The oxytetracycline and tetracycline mainly adsorption on Lolium perenne,alfalfa and couch grass.The tylosin adsorption on Lolium perenne,alfalfa and chicory.The penicillin,chloramphenicol and erythromycin residue on Lolium perenne, alfalfa and clover.The streptomycin residues on Lolium perenne, clover, chicory and bromus inermis.The kanamycin mainly adsorption on Lolium perenne,alfalfa and chicory. The antibiotic residual has the relationship with the amount of adding to the water,and the more addition the higher residual.When add oxytetracyclin200mg to the water,the residual in grass about24.2μg/kg,and the residual is13.2μg/kg when add100mg.When add the same concentration antibiotic to the water,test the residual in forage on different periods and it shows an decline trend.The forage has digestion effect on antibiotics.
(3) The bacteria could produce antibitics itself, and its medium also can find antibiotics.The herbs are irrigated with the bacteria could be detected antibiotics residue.
引文
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