1、F;己中华人民共和国进出口商品检验行业标准SN 0296-93 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 monensin residues in poultry meat for export -Bioautography method 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SN 0296- 93 Method for the determination of monensin residues in
2、 poultry meat for export -Bioautography method 1 主题内容与适用范围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藏留量的检验。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，件最低抽样数，件125 l 26100 5 101 250 10 251 500 15 5011 000 17 1 0012 500 20 2.3 抽样方法按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
3、品，原始样品总量不少于2埠，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，充分混匀，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 样品保存将试样于一18CC冷冻保存。注2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
4、.1 微量注射器:50L。SN 0296- 93 3- 2. 2 薄层板:硅胶，Q/YT257-85SG , 5 cmX20 cm，使用前1l0C活化2h。3. 2. 3 展开缸:240mmX57 mmX32 mmo 3. 2. 4 游标卡尺:测量范围0200mm，精度0.02mm或使用抑菌圈测量仪测量。3.2.5 离心机:转速3000 r/mino 3.2.6 旋转撒缩器。3. 2. 7 恒温培养箱:37士1CC。3.2.8 高压灭菌器。3. 2. 9 长方形培养皿:20 cm X 10 cm X 5 cm 0 3. 2. 10 均质器:带均质杯250mL。3.3 试剂和培养基3. 3. 1
5、 试剂3. 3. 1. 1 甲醇:分析纯。3.3.1.2 四氯化碳:分析纯。3. 3. ,. 3 乙酸乙醋:分析纯。3. 3. .4 莫能菌素标准品:960g(效价)/mg(中国兽药监察所提供)。3.3.1. 5 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供)。3.3. 2 培养基3.3.2. , 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 肉汤培字号基(见附录A第A3章)。3. 4 测定步骤3. 4. , 工作液制备3. 4. 莫能菌素标准贮备液准确称
6、取适量的莫能菌素标准品，用甲醇溶解配制成r&.度为500!-.(g(效价)/mL的莫能菌素标准帘模。配制后于冰箱中保存，一周内使用。3. 4. 1. 2 莫能菌素标准工作攘吸取标准贮备液，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。以上稀释液均须当日配制。3. 4. 1. 3 菌种培养及芽胞菌悬浮液制备将菌种安音在瓶的上部消毒后敲碎，加入少量肉汤培养基，使其溶解井移至肉汤管中混匀。置37土1C温箱中培养24h。将培养物接种子菌种培养基中，置于37士1C培养一周，镜检含芽胞菌数达85%以上，便可制备芽胞悬浮准。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
7、中，充分摇匀后，于3000 r/min离心30 min，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65C水浴中加热30min，然后，于1000 r/min离心5min，取上清液并转入灭菌试管中，即为芽胞菌悬浮液，百于冰箱中保存，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3000 r/min离心20min。取其上清液15mL，转入盛有5mL蒸馆水的250mL分液漏斗中，1昆合后加入10mL四氯化碳，充分振摇，静置分层，放出四氧化服层于150mL茄形
8、瓶中。用四2 SN 0296- 9 3 氧化碳再重复提取两次，合并四氯化碳至上述茄形瓶中，于5060C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。此样被中相当于禽肉试样放度为15g/mLo 3. 4.3 测定3. 4. 3. 1 生物自显影将每个样品及标准洛液分别点在四块薄层板I二，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20L试液和3g(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醋进行展开，直至榕剂前沿线距板顶端1.5 cm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
9、作将己熔化并冷却至5055C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士lC培养18ho 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固的品值(约O.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榕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(20 L)的抑菌圈直径近似(直径相差不超过士1mm)，则其样品中莫能
10、菌素含量按下式计算:式中:x-一样品中莫能菌素的含量，mg/kg;x=三l C一试验中所用的标准榕液的浓度，问/mL;m 最终试液所代表的禽肉试样的改度，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)/悔。4.2 回收率回收率的实验数据:莫能菌素放度在O.200. 67 mg(效价)/kg范围内，回收率为81%100%。3 Al 菌种用培养基蛋白陈牛肉浸膏氧化饷琼脂蒸锢水SN 0296-93 附录A培养基成分及制备方法(补充件)10.0 g 5.0 g 2. S g 15.0 g 1 000 mL 将上述各成分于蒸悟水中加热溶解，用1mol/L氢氧化纳榕掖或
11、10%盐酸调节pH，使其灭菌后pH为6.5士O.1，分装于试管或克氏瓶内.于121C 15 min I:j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10.0 g 酵母浸膏2.5 g 葡萄糖10.0 g 琼脂15.0 g 蒸悟水1 000 mL 将上述各成分于蒸锢水中加热洛解，用1mol/L氢氧化纳溶液或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121cC 15 min高压灭菌，备用。A3 肉汤培养基蛋白陈10.0 g 牛肉浸膏5.0 g 氧化纳2.5 g 蒸榴水1 000 mL 将上述各成分于蒸馆水中加热溶解，用1mol/L氢氧化纳榕液或10%
12、盐酸调节pH，使其灭菌后pH为7.0土0.1，分装于试管中，于121C15min高压灭菌。4 附加说明:试样(10.0g)SN 0296-93 附录B检验程序图(补充件)于均质杯内加入20 mL甲醇均质2mn 移入离，心管离心20min(3000r / min) 取上清液(15mL) l 5 mL蒸馆水的分液漏斗中加入四氯化碳(10mL) ! 充分振叫叫层转入茄形瓶中，再重复两次合并入上述茄形瓶中蒸干用0.5mL甲醇溶解层析培养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
13、用培养基本标准等同采用日本厚生省检验方法:畜水产食品中残留物质检查法(1990)0 5 Professional Standard of the People s Republic of China for Import and Export Commodity Inspection Method for the determination of monensin residues in poultry meat for export - Boautography method 1 Scope and field of application SN 0296-93 This standard
14、specifies the method of samp1ing ,samp1e preparation and determination by bioauto graphy method of monensin residues in poultry meat for export. This standard is app1icab1e to the de1 errnination of rnonensin residues in chicken rneat for export. 2 Sample and sample preparation 2. 1 lnspection 10t T
15、he quantity of an inspection 10t shou1d not be rnore than 2 500 packages. The characteristics of the cargo within the same inspection 10t , such as packing , rnark , origin , specification and grade , shou1d be the same. 2. 2 Quantity of sarnp1e taken Nurnber of packages Minirnum number of in each i
16、nspection 10t packages to be taken 1-25 1 26一1005 101-250 10 251-500 15 501-1 000 17 1 001-2 500 20 2.3 Samp1ing procedure A nurnber of packages specified in 2. 2 are taken at randorn and opened one by one. From each at 1east one bag sha11 be taken as prirnary samp1e. The tota1 weight of a11 prirnar
17、y sarnp1es shou1d not be 1ess than 2 kg. which sha11 be p1aced in a clean container ,sea1ed ,1abe1ed and sent to 1aboratory in tirne. 2.4 Preparation of test sarnp1e Part of representative sarnp1e is taken from each bag of the primary samp1e , the edib1e portions are homogenized by grinding in a mea
18、t grinder. The homogemzed samp1e is thoroughtly mixed and reduced to at 1east 1 kg by quartering as test samp1e. The test sample is placed in a clean container which shall Approved by the State Administrition of Import and Export Commodity Inspection of the People s Republic of China on Dec. 28 , 19
19、93 1) Implemented from May. 1 .1994 SN 0296- 93 be sealed and labeled. 2. 5 Storage of test sample The test sample should be frozen and stored at -18C. Note: In the course of sampling and sample preparation , precaution must be taken to avoid contamination or any factor which may cause the change of
20、 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 chro matographed by thin layer ch
21、romotography and determined by bioautography method. 3. 2 Apparatus and equipments 3. 2. 1 Micro-syringe: 50L 3. 2. 2 Thin-layer plate:Silica gel ,Q/YT 257-85SG , 5 cmX 20 cm, activate at 1l0C for 2 h before use. 3. 2. 3 Developing tank: 240 mm X 57 mm X 32 mm. 3. 2. 4 Vernier calliper:measuring ran
22、ge 0-200 mm ,precision 0.02 mm or use measurer. 3.2. 5 Centrifuge: 3 000 r /min. 3.2.6 Rotaryevaporator. 3.2.7 Incubator:37士1C. 3. 2. 8 Autoclave sterilizer. 3.2.9 Rectanguler culture dish:20 cmX 10 cmX 5 cm. 3. 2.10 Homogenizer:with homogeneous cup (250 mL). 3. 3 Reagents and media 3. 3. 1 Reagents
23、 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. 4 Monensin standard: 960g (potency) /mg (Provided by Veterinary Drug Supervision Insti tute of China) 3. 3. 1. 5 Bacterial strain Bacillus subtilis A TCC 66
24、33 (Provided by Drug and Biological Product Inspection Institute of the State Ministry of Public Health). 3. 3. 2 Media 3.3.2.1 Medium for strain culture (See Appendix A l). 3. 3. 2. 2 Midium for bioautography (See Appendix A2). 3. 3. 2. 3 Broth medium(See Appendix A3). 3.4 Procedure of determinatio
25、n 3. 4. 1 Preparation of working solution 3.4. 1. 1 Monensin standard stock solution: Accurately weigh a proper quantity of monensin standard and dissolve in methanol to prepare 500 /Lg(potency) /mL standard stock solution. Store in a refrigera tor , which can be used within a week. 3. 4. 1. 2 Monen
26、sin standard working solution 7 SN 0296 93 Pipet a certain amount of monensin standard stock solution , and dilute with methanol to prepare 2 ,3.4.5 , 10 , and 15g (potency) /mL standard working solutions respectively , a11 of above solution should be preparaed a t the same day. 3.4. ,. 3 Culture of
27、 strain and preparation of spores suspension After 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 we11 and incu bate at 37士1C for 24 h. Transfer the ncubated culture
28、to medium for strain and incubate at 37士1Cfor a week. When the number of spores detected by microscope exceed 85 % , it can be used for the preparation of spores suspension. Wash down the spores with sterilized saline and transfer to a centrifugal tube , after thoroughly mixing ,centrifuge at 3 000
29、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 a 65C water bath for 30 min. Centrifuge again at 1 000 r /min for 5 min. Transfer the supernate (the spores suspension) into
30、 sterile tubes. The spores suspension can be used within a month by storing in a refrigera tor. 3. 4. 2 Preparation of sample solution Accuately weigh 10.0 g of the chopped sample(accurate to 0.1 g) into a homogeneous cup. Add 20 mL of methanol and homogenize for 2 min. Transfer it into a centrifuga
31、l tube and centrifuge at 3 000 r /min for 20 min. Pipet 15 mL of supernate into a 250 mL separatory funnel containing 5 mL of disti11ed water .after mixing ,add 10 mL of carbon tetrachloride and shake vigorously. Let stand to sep arate and drain carbon tetrachloride into a 150 mL Mojonner-type flask
32、. Repeat this extraction step twice using carbon tetrachloride and combine these carbon tetrachloride into the above Mojonner-type flask. Evaporate the carbon tetrachloride to dryness in a water bath at 50-60cC with a rotary evapora tor , add O. 5 mL of methanol to dissolve the residue for TLC. In t
33、his solution, the concentration of poultry meat sample is equivalent to 15 g/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 a baseline across the plate 2 cm from the bottorn , then 5pot 20L sample e
34、xtract and monensin standard solutionC3g(potency) /mL) on the baseline , the sample spot shall be 2.5 cm away from thc standard spot. Develop the TL plate in the tank using ethyl acetate as developing solution , until the front mar gin of the solvent is 1. 5 cm from the top of plate. Remove the plat
35、e , after the plate is air-dried , it is ready for later incubation. Place the thin-layer plate into a sterilized rectanguler culture dish horizontally. Aseptica11y spray the melted and cooled to 50-55 C medium for bioautography onto the surface of the plate , then pour 10 mL of the above medium ino
36、culated with BacilLus subtilis spores suspenson over the surface of the plate. Allow the plate to keep on level surface untl the agar solidifies and incubate at 37 C for 18 h. 3. 4. 3. 2 Contrast test The operation of the contrast test is the ame as that describe in the method of determination , but
37、 with omission of sample addition. 3. 5 Calculation and expresson of result After incubation , the test is considered positive if the sample shows inhibit zone on the plate wth the ,-ame Rf value(ca 0.38) , by comparing with the inhibit zone of the monesin standard solution. 8 E SN 0296-93 Vhen the
38、sample is showed positive result ,measure the diameters of the positive inhibit zone on four plates ,cause by the sample extraction and standard solution , calculate the averages respectvely. When the relative standard deviation(RSD) values of each group are not more than 5% ,and the diam eters of i
39、nhibit zone of sample extraction is about the same(within士1mm)as that of monensin stan dard solution(20L) ,then the content of monensin can be calculated according to the equation below: where x=!:_ m X -content of monensin in sample , mg/kg ; C concentration of standard solution which is used in th
40、e test，问/mL;m-corresponding mass of the poultry meat in each milliliter of the fnal test solution ,g/mL. 4 Limit of determnation and recovery 4. 1 Limit of determination The limit of determinaton is 0.20 mg (potency) /kg. 4. 2 Recovery According to the experimental data ,when the fortifying concentr
41、ation of monensin is in the range of 0.20-0.67 mg(potency)/kg,the recovery is 81%-100%。9 A1 SN 0296- 93 Appendix A Ingredients of medium and preparation method CSupplement) Medium for strain culture Peptone 10.0 g Beef extract 5.0 g NaCl 2.5 g Agar 15.0 g Distilled water 1 000 mL Dissolve each ingre
42、dients in distilled water. After heating and stirring ,adjust the pH with sodium hydroxide solution (1 mol/L)or hydrochloric acid (10%) so that the value after autoclaving is 6.5 士o.1. Dispense into test tubes or Kolle flask. Autoclave at ll-C for 15 min ,and prepare stant as re quired. A2 Medium fo
43、r bioautography Peptone Yeast extract Glucose Agar Distilled water 10.0 g 2.5 g 10.0 g 15.0 g 1000 mL Dissolve each ingredients in distilled water. After heating and stirring ,adjust the pH with sodium hydroxide solution (1 mol/L) or hydrochioric acid (10%) so that the value after autoclaving is 6.
44、0 士O.1. Dispense into conical flasks ,autoclave at 121C for 15 min. Store for later use. A3 roth medium Peptone 10.0 g Beef extract 5.0 g NaCl 2.5 g Distilled water 1 000 mL Dissolve each ingredients in distilled water. After heating and stirring ,adjust the pH with sodium hydroxide solution (1 mol/
45、L) or hydrochloric acid (10 %) so that the value after autoclaving is 6. 5 土0.1.Dispense into tubes. Autoclav at 121 C for 15 min. 10 SN 029693 Appendix B Procedure of test CSupplement) sample (1 0. Og) add 20 mL methanul into homogeneous cup homogenize for 2 min transfcr into a centrifugal tubc cen
46、trifuge for 20 min(3000 r / min) pipet the supernate(l5 mL) transfer into a separatory funnel with 5 mL DW add carbon tetrachloride(lO mL) stir thoroughly and drain the layer of carbon tetrachloride into a Monnojier- typc flask ,repeat twicc combine them into the same Monnojier- type flask s s e n V
47、J r d o t ilelei- t 到ur o nr au v e dissolve the residues with O. 5 mL mehtanol chromatography incubation determination cxprCSSlon of rcsu1t incubation of test strain preparation of strain solution medium for bioautography 11 SN 0296-93 Additio乓alexplanations: This standard was proposed by the State
48、 Administration of Import and Export Commodity In spection Bureau of the People s Republic of China. This standard was drafted by the Tianjin Import and Export Commodity Inspection Bureau of the People s Republic of China. This standard was mainly drafted by Yuan Ersen , Wang Suqing , Li Jianying. This standard is identical with th巳determinationmethod which is passed by The Public Health Department of Japan:畜水产食品中町残留物质检查法(1990).12 何1NO(京)新登字023号z m 中国标准出版社秦皇岛印刷厂印刷1994年12月第一版书号:155066 2-9574 1994年12月第一次印刷中国标准出版社出版
