【SN商检标准】 出口禽肉中莫能菌素残留量检验方法生物自显影法

中华人民共和国进出口商品检验行业标准SN0296-93
上海市技术监督情学研究
登号Q7966143
出口禽肉中莫能菌素残留量检验方法生物自显影法
Method for the determination of monensinresidues in poultry meat for exportBioautographymethod
1993-12-28发布
1994-05-01实施
，中华人民共和国国家进出口商品检验局发布中华人民共和国进出口商品检验行业标准出口禽肉中莫能菌素残留量检验方法生物自显影法
Method for the determination of monensinresidues in poultry meat for export-Bioautography method
1主题内容与适用范围
SN 0296-93
本标准规定了出口禽肉中莫能菌素残留量的抽样、制样和生物自显影测定法。本标准适用于出口冻鸡中莫能菌素残留量的检验。2抽样和制样
2.1检验批
以不超过2500件商品为一检验批，同一检验批的商品应具有相同特征，如包装、标记、产地、规格和等级等。2.2抽样数量
批量，件
26~100
101～250
251~500
501~1000
1001～2500
2.3抽样方法
最低抽样数，件
按2.2规定的抽样件数随机抽取，逐件开启。每件至少取一袋作为原始样品，原始样品总量不少于2kg，放入清洁容器内，加封后标明标记，及时送交实验室。2.4样品制备
从每袋原始样品中取出部分有代表性样品，将可食部分放入高速组织捣碎机中捣碎均匀，充分混勾，用四分法缩分出不少于1kg试样。装入清洁容器内，加封后标明标记。2.5样品保存
将试样于一18℃冷冻保存。
注：在抽样及制样过程中，必须防止样品受到污染或发生残留物含量的变化。3测定方法
3.1方法提要
用甲醇提取肉样中莫能菌素，经四氯化碳萃取后浓缩至干，以甲醇溶解残留物，用薄层分离，再用生中华人民共和国国家进出口商品检验局1993-12-28批准1994-05-01实施
物自显影法测定。
3.2设备和材料
3.2.1微量注射器：50μL。
SN0296-93
3.2.2薄层板：硅胶，Q/YT257-85SG，5cm×20cm，使用前110C活化2h3.2.3展开缸：240mm×57mm×32mm。3.2.4游标卡尺：测量范围0~200mm，精度0.02mm或使用抑菌圈测量仪测量。3.2.5离心机：转速3000r/min。3.2.6旋转浓缩器。
3.2.7恒温培养箱：37土1℃。
3.2.8高压灭菌器。
3.2.9长方形培养皿：20cm×10cm×5cm。3.2.10均质器：带均质杯250mL。3.3试剂和培养基
3.3.1试剂
3.3.1.1甲醇：分析纯。
3.3.1.2四氯化碳：分析纯。
3.3.1.3乙酸乙酯：分析纯。
3.3.1.4莫能菌素标品：960μg（效价）/mg（中国兽药监察所提供）。试验菌种：枯草杆菌（Bacillussubtilis），菌种号ATCC6633（卫生部药品生物制品检定所提3.3.1.5
供）。
3.3.2培养基
3.3.2.1菌种用培养基（见附录A第A1章）。3.3.2.2生物自显影用培养基（见附录A第A2章）。3.3.2.3肉汤培养基（见附录A第A3章）。3.4测定步骤
3.4.1工作液制备
3.4.1.1莫能菌素标准贮备液
准确称取适量的莫能菌素标准品，用甲醇溶解配制成浓度为500g（效价）/mL的莫能菌素标准溶液。配制后于冰箱中保存，一周内使用。3.4.1.2莫能菌素标准工作液
吸取标准贮备液，用甲醇分别稀释成2,3，4，5，10，和15g（效价）/mL的标准工作标准液。以上稀释液均须当目配制。
3.4.1.3菌种培养及芽胞菌悬浮液制备将菌种安颜瓶的上部消毒后敲碎，加入少量肉汤培养基，使其溶解并移至肉汤管中混匀。置37土1℃温箱中培养24h。将培养物接种于菌种培养基中，置于37士1℃培养一周，镜检含芽胞菌数达85%以上，便可制备芽胞悬浮液。
用适量灭菌生理盐水冲洗菌苔，然后将该菌液转移至离心管中，充分摇匀后，于3000r/min离心30min.弃去上清液，再加入同样量的灭菌生理盐水重复离心一次。弃去上清液，再加入适量灭菌生理盐水，摇匀后于65℃水浴中加热30min，然后，于1000r/min离心5min，取上清液并转入灭菌试管中，即为芽胞菌悬浮液，置于冰箱中保存，可使用一个月。3.4.2试液制备：准确称取绞碎试样10g（精确至0.1g）于均质杯中，加入20mL甲醇，均质2min后移入离心管中，于3000r/min离心20min。取其上清液15mL，转入盛有5mL蒸馏水的250mL分液漏斗中，混合后加入10mL四氯化碳，充分振摇，静置分层，放出四氯化碳层于150mL茄形瓶中。用四2
SN0296-93
氯化碳再重复提取两次，合并四氯化碳至上述茄形瓶中，于50～60℃水浴中用旋转蒸发器浓缩至干，加入0.5mL甲醇溶解残留物供TLC实验用。此样液中相当于禽肉试样浓度为15g/mL。3.4.3测定
3.4.3.1生物自显影
将每个样品及标准溶液分别点在四块薄层板上，先于每块薄层板下端2cm处划一条基线，然后在这条基线上分别点20μL试液和3μg（效价)/mL莫能菌素标准溶液，试液和标准溶液点相距2.5cm。在展开缸中用乙酸乙酯进行展开，直至溶剂前沿线距板顶端1.5cm处为止，取出薄层板，风干后备培养用。
将薄层板水平置于高压灭菌的长方形培养皿中，无菌操作将已熔化并冷却至50～55℃的生物自显影用培养基均匀地喷在其表面上，然后用10mL上述接种芽胞菌悬液的培养基铺满整个薄层板，保持水平，待其凝固，于37土1℃培养18h。3.4.3.2对照试验
除不加试样外，按上述测定步骤进行。3.5结果计算和表述
经过培养后，在薄层板上与莫能菌素标准液产生抑菌圈的R：值（约0.38）相同位置上，显现抑菌圈者即为阳性。
当样品判定为阳性时，测量四块薄层板上的试液及标准溶液产生的抑菌圈直径，分别计算出平均值。当每组四个直径值的相对标准偏差（RSD)均不超过5%时，并且试液的抑菌圈直径与标准溶液（20μL)的抑菌圈直径近似（直径相差不超过土1mm），则其样品中莫能菌素含量按下式计算：X=
式中：X-—样品中莫能菌素的含量，mg/kgc——试验中所用的标准溶液的浓度+μg/mLm——最终试液所代表的禽肉试样的浓度，g/mL。4测定低限、回收率
4.1测定低限
本方法测定低限为0.20mg（效价）/kg。4.2回收率
回收率的实验数据：莫能菌素浓度在0.20～0.67mg（效价）/kg范围内，回收率为81%～100%。A1
菌种用培养基
蛋白陈
牛肉浸膏
氮化钠bZxz.net
蒸馏水
SN0296—93
附录A
培养基成分及制备方法
（补充件）
1000mL
将上述各成分于蒸馏水中加热溶解，用1mol/L氢氧化钠溶液或10%盐酸调节pH，使其灭菌后pH为6.5士0.1，分装于试管或克氏瓶内，于121C15min高压灭菌，灭菌后制备成所需斜面备用。A2
生物自显影用培养基
蛋白陈
酵母浸膏
葡萄糖
蒸馏水
1000mL
将上述各成分于蒸馏水中加热溶解，用1mol/L氢氧化钠溶液或10%盐酸调节pH，使其灭菌后pH为6.0士0.1，分装于锥形瓶内，于121℃15min高压灭菌，备用。A3·肉汤培养基
蛋白陈
牛肉浸膏
氯化钠
蒸馏水
1000ml
将上述各成分于蒸馏水中加热溶解，用1mol/L氢氧化钠溶液或10%盐酸调节pH，使其灭菌后pH为7.0士0.1，分装于试管中，于121℃15min高压灭菌。附加说明：
试样（10.0g）
均质2min
SN0296-93
附录B
检验程序图
（补充件）
于均质杯内加入
20mL甲醇
移人离心管
离心20min(3000r/min）
取上清液（15mL）
转入盛有5mL蒸馏
水的分液漏斗中
加入四氯化碳（10mL）
充分振摇后分取四氮化碳层
转入茄形瓶中，再重复两次
合并入上述茄形瓶中
用0.5mL甲醇溶解
结果和表述
本标准由中华人民共和国国家进出口商品检验局提出。本标准由中华人民共和国天津进出口商品检验局负责起草。本标准主要起草人袁而森、王素琴、李剑影。试验菌培养
菌液制备
生物自显影用培养基
本标准等同采用日本厚生省检验方法：畜水产食品中の残留物质检查法（1990)。5
Professional Standard of the People's Republic of ChinaforImportandExportCommodityInspectionMethod for the determination of monensinresidues in poultry.meat for export-Bioautography method
1 Scope and field of applicationSN0296-93
This standard specifies the method of sampling ,sample preparation and determination by bioauto-graphy method of monensin residues in poultry meat for export.This standard is applicable to the determination of monensin residues in chicken meat for export.2 Sample and sample preparation2.1 Inspection lot
The quantity of an inspection lot should not be more than 2 5o0 packages.The characteristics of the cargo within the same inspection lot, such as packing, mark,origin,specification and grade,should be the same.2.2 Quantity of sample takenNumber of packages
in each inspection lot
1—25
26—100
101—250
251-500
501-1000
1001—2500
2.3Sampling procedure
Minimum number of
packages to be taken
A number of packages specified in 2. 2 are taken at random and opened one by one. From each atleast one bag shall be taken as primary sample. The total weight of all primary samples should not beless than 2 kg ,which shall be placed in a clean container,sealed,labeled and sent to laboratory in time.2.4Preparation of test samplePart of representative sample is taken from each bag of the primary sample,the edible portions arehomogenized by grinding in a meat grinder. The homogenized sample is thoroughtly mixed and reducedto at least 1 kg by quartering as test sample. The test sample is placed in a clean container which shallApproved by the State Administrition of Importand Export Commodity Inspection of the People'sRepublic of China on Dec. 28,19936
Implemented from May.1.1994
be sealed and labeled.
2. 5 Storage of test sample
SN0296-93
The test sample should be frozen and stored at -18'℃.Note: In the course of sampling and sample preparation,precaution must be taken to avoid contamination'or anyfactor which may cause the change of residue content.3 Method of determination
3.1 Principle of method
The monensin is extracted from the meat tissues with methanol,partitioned into carbon tetrachlo-ride. After evaporation to dryness,the residues are dissolved in methanol. The prepared extract is chromatographed by thin layer chromotography and determined by bioautography method.3.2 Apparatus and equipments3.2.1 Micro-syringe:50 μL.
3.2.2 Thin-layer plate: Silica gel,Q/YT 257-85SG,5 cm X 20 cm,activate at. 110C for 2 h beforeuse.
3.2.3Developing.tank:240 mmX57mmX32mm.3.2.4 Vernier calliper:measuring range 0200 mm.precision 0. 02 mm or use measurer.3.2.5 Centrifuge:3000 r/min
3.2.6 Rotary evaporator.
3.2.7Incubator:37±1℃.
3.2.8 Autoclave sterilizer.
3. 2. 9 Rectanguler culture dish:20 cm X10 cmX5 cm3.2.10Homogenizer:with homogeneous cup(250mL).3.3 Reagents and media
3.3.1 Reagents
3.3.1.1 Methanol.Analytical grade.3. 3. 1.2 Carbon tetrachloride :Analytical grade.3.3.1.3 : Ethyl acetate :Analytical grade.3.3.1.4Monensin standard:960 μg(potency)/mg (Provided by Veterinary Drug Supervision Insti-tute of China)
3.3.1.5Bacterial strain
Bacillus subtilis ATCC 6633(Provided by Drug and Biological Product Inspection Institute of theState Ministry of Public Health).3.3.2Media
3.3.2.1Mediumforstrainculture(SeeAppendixA1).3.3.2.2Midiumforbioautography(SeeAppendixA2).3.3.2.3Broth medium(See Appendix A3).3. 4 Procedure of determination3.4.1 Preparation of working solution3. 4. 1.1 Monensin standard stock solution :Accurately weigh a proper quantity of monensin standardand dissolve in methanol to prepare 500 μg(potency)/mL standard stock solution.Store in a refrigera-tor,which can be used within a week.3.4.1.2 Monensin standard working solutionSN0296-93
Pipet a certain amount of monensin standard stock solution ,and dilute with methanol to prepare2,3,4,5,10,and 15 μg(potency)/mL standard working solutions respectively,all of above solutionshould be preparaed at the same day.3.4.1.3 Culture of strain and preparation of spores suspensionAfter sterilizing the ampoule of the strain ,cut off the top and add a small amount of broth medi-um in it to dissolve the content. Transfer into the above mentioned broth test-tube ,mix well and incu-bate at 37±1C for 24 h. Transfer the incubated culture to medium for strain and incubate at 37±iCfor a week. When the number of spores detected by microscope exceed 85%,it can be used for thepreparation of spores suspension.Wash down the spores with sterilized saline and transfer to a centrifugal tube,after thoroughlymixing ,centrifuge at 3 000 r/min for 30 min. Discard the supernate and repeat above step once more.Discard the supernate add a certain amount of sterilized saline and mix well,heat the suspension in a65'C water bath for 30 min. Centrifuge again at 1 000 r/min for 5 min. Transfer the supernate (thespores suspension) into sterile tubes. The spores suspension can be used within a month by storing ina refrigerator.
3.4.2Preparationof samplesolutionAccuately weigh 1o. O g of the chopped sample(accurate to O. l g) into a homogeneous cup. Add20 mL of methanol and homogenize for 2 min. Transfer it into a centrifugal tube and centrifuge at3 000 r/min for 20 min.Pipet 15 mL of supernate into a 250 mL separatory funnel containing 5 mL ofdistilled water,after mixing +add 10 mL of carbon tetrachloride and shake vigorously. Let stand to sep-arate and drain carbon tetrachloride into a 15o mL Mojonner-type flask. Repeat this extraction steptwice using carbon tetrachloride and combine these carbon tetrachloride into the above Mojonner-typeflask. Evaporate the carbon tetrachloride to dryness in a water bath at 50-6o'C with a rotary evaporator,add o. 5 mL of methanol to dissolve the residue for TLC. In this solution, the concentration ofpoultrymeatsampleis equivalentto15g/mL.3.4.3 Determination
3.4.3.1 Bioautography
Spot each sample and standard solution on four plates respectively. On each plate,at first scribe abaseline across the plate 2 cm from the bottom,then spot 2o μL sample extract and monensin standardsolution (3 μg(potency)/mLJ on the-baseline,the sample spot shall be 2. 5 cm away from the standardspot. Develop the TL plate in the tank using ethyl acetate as developing solution,until the front mar-gin of the solvent is l. 5 cm from the top of plate. Remove the plate,after the plate is air-dried,it isready for later incubation.
Place the thin-layer plate into a sterilized rectanguler culture dish horizontally. Aseptically spraythe melted and cooled to 50-55'C medium for bioautography onto the surface of the plate,then pourlo mL of the above medium inoculated with Bacillus subtilis spores suspension over the surface of theplate. Allow the plate to keep on level surface until the agar solidifies and incubate at 37C for 18 h.3.4.3.2Contrast test
The operation of the contrast test is the same as that describe in the method of determination,butwith omission of sample addition.3. 5 . Calculation and expression of resultAfter incubation,the test is considered positive if the sample shows inhibit zone on the plate withthe same R value(ca O. 38),by comparing with the inhibit zone of the monesin standard solution.8
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