标准搜索结果: 'GB 4789.12-2016英文版'
| 标准号码 | 内文 | 价格(元) | 第2步 | 交付天数[PDF] | 标准名称 | 相关标准 |
| GB 4789.12-2016 |
英文版
| 300 |
购买全文
|
现货, 9秒内下载
|
食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验
|
GB 4789.12-2016
|
标准编号: GB 4789.12-2016 (GB4789.12-2016)
中文名称: 食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验
英文名称: National Food Safety Standard -- Food Microbiological Examination -- Clostridium Botulinum and Botulinum Toxin
行业: 国家标准
中标分类: X09
字数估计: 13,165
发布日期: 2016-12-23
实施日期: 2017-06-23
旧标准 (被替代): GB/T 4789.12-2003
标准依据: National Health and Family Planning Commission Notice No.17 of 2016
GB 4789.12-2016: 食品安全国家标准 食品微生物学检验 肉毒梭菌及肉毒毒素检验
GB 4789.12-2016 英文名称: National Food Safety Standard -- Food Microbiological Examination -- Clostridium Botulinum and Botulinum Toxin
1 范围
本标准规定了食品中肉毒梭菌及肉毒毒素的检验
方法。
本标准适用于食品中肉毒梭菌及肉毒毒素的检验。
2 设备和材料
除微生物实验室常规灭菌及培养设备外,其他设备和材料如下:
2.1 冰箱:2℃~5℃、-20℃。
2.2 天平:感量0.1g。
2.3 无菌手术剪、镊子、试剂勺。
2.4 均质器或无菌乳钵。
2.5 离心机:3000r/min、14000r/min。
2.6 厌氧培养装置。
2.7 恒温培养箱:35℃±1℃、28℃±1℃。
2.8 恒温水浴箱:37℃±1℃、60℃±1℃、80℃±1℃。
2.9 显微镜:10倍~100倍。
2.10 PCR仪。
2.11 电泳仪或毛细管电泳仪。
2.12 凝胶成像系统或紫外检测仪。
2.13 核酸蛋白分析仪或紫外分光光度计。
2.14 可调微量移液器:0.2μL~2μL、2μL~20μL、20μL~200μL、100μL~1000μL。
2.15 无菌吸管:1.0mL、10.0mL、25.0mL。
2.16 无菌锥形瓶:100mL。
2.17 培养皿:直径90mm。
2.18 离心管:50mL、1.5mL。
2.19 PCR反应管。
2.20 无菌注射器:1.0mL。
2.21 小鼠:15g~20g,每一批次试验应使用同一品系的KM或ICR小鼠。
3 培养基和试剂
除另有规定外,PCR试验所用试剂为分析纯或符合生化试剂标准,水应符合GB/T 6682中一级水
的要求。
3.1 庖肉培养基:见A.1。
3.2 胰蛋白酶胰蛋白胨葡萄糖酵母膏肉汤(TPGYT):见A.2。
3.3 卵黄琼脂培养基:见A.3。
3.4 明胶磷酸盐缓冲液:见A.4。
3.5 革兰氏染色液:见A.5。
3.6 10%胰蛋白酶溶液:见A.6。
3.7 磷酸盐缓冲液(PBS):见A.7。
3.8 1mol/L氢氧化钠溶液。
3.9 1mol/L盐酸溶液。
3.10 肉毒毒素诊断血清。
3.11 无水乙醇和95%乙醇。
3.12 10mg/mL溶菌酶溶液。
3.13 10mg/mL蛋白酶K溶液。
3.14 3mol/L乙酸钠溶液(pH5.2)。
3.15 TE缓冲液。
3.16 引物:根据表1中序列合成,临用时用超纯水配制引物浓度为10μmol/L。
4 检验程序
肉毒梭菌及肉毒毒素检验程序见图1。
5 操作步骤
5.1 样品制备
5.1.1 样品保存
待检样品应放置2℃~5℃冰箱冷藏。
5.1.2 固态与半固态食品
固体或游离液体很少的半固态食品,以无菌操作称取样品25g,放入无菌均质袋或无菌乳钵,块状
食品以无菌操作切碎,含水量较高的固态食品加入25mL明胶磷酸盐缓冲液,乳粉、牛肉干等含水量低
的食品加入50mL明胶磷酸盐缓冲液,浸泡30min,用拍击式均质器拍打2min或用无菌研杵研磨制
备样品匀液,收集备用。
5.1.3 液态食品
液态食品摇匀,以无菌操作量取25mL检验。
5.1.4 剩余样品处理
取样后的剩余样品放2℃~5℃冰箱冷藏,直至检验结果报告发出后,按感染性废弃物要求进行无
害化处理,检出阳性的样品应采用压力蒸汽灭菌方式进行无害化处理。
5.2 肉毒毒素检测
5.2.1 毒素液制备
取样品匀液约40mL或均匀液体样品25mL放入离心管,3000r/min离心10min~20min,收集
上清液分为两份放入无菌试管中,一份直接用于毒素检测,一份用于胰酶处理后进行毒素检测。液体样
品保留底部沉淀及液体约12mL,重悬,制备沉淀悬浮液备用。
胰酶处理:用1mol/L氢氧化钠或1mol/L盐酸调节上清液pH至6.2,按9份上清液加1份10%
胰酶(活力1∶250)水溶液,混匀,37℃孵育60min,期间间或轻轻摇动反应液。
5.2.2 检出试验
用5号针头注射器分别取离心上清液和胰酶处理上清液腹腔注射小鼠3只,每只0.5mL,观察和
记录小鼠48h内的中毒表现。典型肉毒毒素中毒症状多在24h内出现,通常在6h内发病和死亡,其
主要表现为竖毛、四肢瘫软,呼吸困难,呈现风箱式呼吸、腰腹部凹陷、宛如峰腰,多因呼吸衰竭而死亡,可初步判定为肉毒毒素所致。若小鼠在24h后发病或死亡,应仔细观察小鼠症状,必要时浓缩上清液
重复试验,以排除肉毒毒素中毒。若小鼠出现猝死(30min内)导致症状不明显时,应将毒素上清液进
行适当稀释,重复试验。
注:毒素检测动物试验应遵循GB 15193.2《食品安全国家标准 食品毒理学实验室操作规范》的规定。
5.2.3 确证试验
上清液或(和)胰酶处理上清液的毒素试验阳性者,取相应试验液3份,每份0.5mL,其中第一份加
沸10min;第三份加等量明胶磷酸盐缓冲液,混匀。将三份混合液分别腹腔注射小鼠各两只,每只
0.5mL,观察96h内小鼠的中毒和死亡情况。
结果判定:若注射第一份和第二份混合液的小鼠未死亡,而第三份混合液小鼠发病死亡,并出现肉
毒毒素中毒的特有症状,则判定检测样品中检出肉毒毒素。
5.2.4 毒力测定(选做项目)
取确证试验阳性的试验液,用明胶磷酸盐缓冲液稀释制备一定倍数稀释液,如10倍、50倍、100倍、
500倍等,分别腹腔注射小鼠各两只,每只0.5mL,观察和记录小鼠发病与死亡情况至96h,计算最低
致死剂量(MLD/mL或 MLD/g),评估样品中肉毒毒素毒力,MLD等于小鼠全部死亡的最高稀释倍数
乘以样品试验液稀释倍数。例如,样品稀释两倍制备的上清液,再稀释100倍试验液使小鼠全部死亡,
5.2.5 定型试验(选做项目)
根据毒力测定结果,用明胶磷酸盐缓冲液将上清液稀释至10MLD/mL~1000MLD/mL作为定
型试验液,分别与各单型肉毒毒素诊断血清等量混合(国产诊断血清一般为冻干血清,用1mL生理盐
水溶解),37℃孵育30min,分别腹腔注射小鼠两只,每只0.5mL,观察和记录小鼠发病与死亡情况至
96h。同时,用明胶磷酸盐缓冲液代替诊断血清,与试验液等量混合作为小鼠试验对照。
结果判定:某一单型诊断血清组动物未发病且正常存活,而对照组和其他单型诊断血清组动物发病
死亡,则判定样品中所含肉毒毒素为该型肉毒毒素。
注:未经胰酶激活处理的样品上清液的毒素检出试验或确证试验为阳性者,则毒力测定和定型试验可省略胰酶激活处理试验。
5.3 肉毒梭菌检验
5.3.1.1 取出庖肉培养基4支和TPGY肉汤管2支,隔水煮沸10min~15min,排除溶解氧,迅速冷
却,切勿摇动,在 TPGY 肉汤管中缓慢加入胰酶液至液体石蜡液面下肉汤中,每支1mL,制备成
TPGYT。
5.3.1.2 吸取样品匀液或毒素制备过程中的离心沉淀悬浮液2mL接种至庖肉培养基中,每份样品接
种4支,2支直接放置35℃±1℃厌氧培养至5d,另2支放80℃保温10min,再放置35℃±1℃厌氧
培养至5d;同样方法接种2支 TPGYT肉汤管,28℃±1℃厌氧培养至5d。
注:接种时,用无菌吸管轻轻吸取样品匀液或离心沉淀悬浮液,将吸管口小心插入肉汤管底部,缓缓放出样液至肉汤中,切勿搅动或吹气。
5.3.1.3 检查记录增菌培养物的浊度、产气、肉渣颗粒消化情况,并注意气味。肉毒梭菌培养物为产气、肉汤浑浊(庖肉培养基中A型和B型肉毒梭菌肉汤变黑)、消化或不消化肉粒、有异臭味。
5.3.1.4 取增菌培养物进行革兰氏染色镜检,观察菌体形态,注意是否有芽胞、芽胞的相对比例、芽胞在
5.3.1.5 若增菌培养物5d无菌生长,应延长培养至10d,观察生长情况。
5.3.1.6 取增菌培养物阳性管的上清液,按5.2方法进行毒素检出和确证试验,必要时进行定型试验,
阳性结果可证明样品中有肉毒梭菌存在。
注:TPGYT增菌液的毒素试验无需添加胰酶处理。
5.3.2 分离与纯化培养
5.3.2.1 增菌液前处理,吸取1mL增菌液至无菌螺旋帽试管中,加入等体积过滤除菌的无水乙醇,混
匀,在室温下放置1h。
5.3.2.2 取增菌培养物和经乙醇处理的增菌液分别划线接种至卵黄琼脂平板,35℃±1℃厌氧培养
48h。
则,在菌落周围形成乳色沉淀晕圈(E型较宽,A型和B型较窄),在斜视光下观察,菌落表面呈现珍珠样
虹彩,这种光泽区可随蔓延生长扩散到不规则边缘区外的晕圈。
5.3.2.4 菌株纯化培养,在分离培养平板上选择5个肉毒梭菌可疑菌落,分别接种卵黄琼脂平板,35℃±
1℃,厌氧培养48h,按5.3.2.3观察菌落形态及其纯度。
5.3.3 鉴定试验
5.3.3.1 染色镜检
挑取可疑菌落进行涂片、革兰氏染色和镜检,肉毒梭菌菌体形态为革兰氏阳性粗大杆菌、芽胞卵圆
形、大于菌体、位于次端,菌体呈网球拍状。
5.3.3.2 毒素基因检测
GB 4789.12-2016
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National food safety standard -
Food microbiological examination -
Clostridium botulinum and botulinum toxin test
ISSUED ON. DECEMBER 23, 2016
IMPLEMENTED ON. JUNE 23, 2017
Issued by. National Health and Family Planning Commission of the PRC;
China Food and Drug Administration.
3. No action is required - Full-copy of this standard will be automatically &
immediately delivered to your EMAIL address in 0~60 minutes.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Equipment and materials ... 4
3 Medium and reagents ... 5
4 Test procedures ... 6
5 Operating procedures ... 7
6 Result report ... 15
Appendix A Medium and reagent ... 16
Foreword
This standard replaces GB/T 4789.12-2003 “Food hygiene microbiological
examination - Clostridium botulinum and botulinum toxin test”.
As compared with GB/T 4789.12-2003, the main changes of this standard are
as follows.
- CHANGE the standard name into “National food safety standard - Food
microbiological examination - Clostridium botulinum and botulinum toxin
test”;
- ADD the PCR identification method;
- ADD the results and reports;
- ADD the Appendix A;
- MODIFY the equipment and materials;
- MODIFY the medium and reagents;
- MODIFY the test procedure;
- NORMALIZE the sample preparation process;
- MODIFY the test methods of enrichment isolation medium part in the
operation procedures.
National food safety standard -
Food microbiological examination -
Clostridium botulinum and botulinum toxin test
1 Scope
This standard specifies the test method for the clostridium botulinum and
botulinum toxin in food.
This standard applies to the test of the clostridium botulinum and botulinum
toxin in food.
2 Equipment and materials
In addition to routine sterilization and culture equipment for microbiological
laboratories, other equipment and materials are as follows.
2.1 Refrigerator. 2 °C ~ 5 °C, -20 °C.
2.2 Balance. sensitivity of 0.1 g.
2.3 Sterile surgical scissors, tweezers, reagent spoon.
2.4 Homogenizer or sterile mortar.
2.5 Centrifuge. 3000 r/min, 14000 r/min.
2.6 Anaerobic culture device.
2.7 Constant temperature incubator. 35 °C ± 1 °C, 28 °C ± 1 °C.
2.8 Constant temperature water bath. 37 °C ± 1 °C, 60 °C ± 1 °C, 80 °C ± 1 °C.
2.9 Microscope. 10 folds to 100 folds.
2.10 PCR instrument.
2.11 Electrophoresis or capillary electrophoresis.
2.12 Gel imaging system or UV spectrophotometer.
2.13 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
food into small pieces; as for the solid food having high water content, ADD 25
mL of gelatin phosphate buffer solution; AND as for the milk powder, beef jerky
and other foods of low water content, ADD 50 mL of gelatin phosphate buffer
solution; MAKE it immersed for 30 min; USE the vibrating homogenizer to beat
it for 2 min or otherwise USE the sterile grinding pestle to grind and prepare
the sample homogenate; COLLECT it to prepare for use.
5.1.3 Liquid food
mL of liquid food for testing.
5.1.4 Remaining sample disposal
WEIGH the remaining sample; REFRIGERATE it in a 2 °C ~ 5 °C refrigerator;
after the test result report is issued, FOLLOW the infectious waste
requirements to perform harmless disposal; AND the sample detected of
positive shall be subjected to harmless disposal by the pressure steam
sterilization method.
5.2 Botulinum toxin detection
5.2.1 Preparation of toxin solution
sample; PLACE it into the centrifuge tube; CENTRIFUGE it at 3000 r/min for
10 min ~ 20 min; COLLECT the supernatant; DIVIDE it into two parts; PLACE it
into the sterile test tube; USE one part for toxin detection and the other part for
toxin detection after trypsin treatment. As for the liquid sample, RETAIN about
12 mL of bottom sediment and liquid; RE-SUSPEND it; PREPARE the
sediment suspension to prepare for use.
Trypsin treatment. USE 1 mol/L sodium hydroxide or 1 mol/L of hydrochloric
acid to adjust the supernatant pH to 6.2; ADD 1 part of 10% trypsin (1.250)
aqueous solution into 9 parts of supernatants; MIX it uniformly; INCUBATE it at
interval.
5.2.2 Detection test
USE No.5 needle syringe to respectively take the centrifuged supernatant and
trypsin treatment supernatant; PERFORM intraperitoneal injection for 3 mice,
0.5 mL for each mouse; OBSERVE and RECORD the poisoning performance
of mice within 48 h. The typical symptoms of botulinum toxin poisoning mostly
appear within 24 hours, the mice usually onset and die within 6 h, AND the
main symptoms include hair erection, limbs weakness and limp, breathing
In accordance with the results of toxicity determination, USE the gelatin
MLD/mL, which is used as the type test sample solution; respectively MIX it
with equal amount of each single type diagnosis serum of botulinum toxin (the
domestic diagnosis serum is generally the frozen dry serum, which is dissolved
by 1 mL of saline); INCUBATE it at 37 °C for 30 min; respectively MAKE
intraperitoneal injection for two mice, 0.5 mL for each mouse; OBSERVE and
RECORD the mice onset and death conditions within 96 h. Meanwhile, USE
the gelatin phosphate buffer to substitute the diagnosis serum; and MIX it with
the same amount of test solution and USE it as the mice test control.
Result judgment. if the animal in a certain single type diagnosis serum group
other single type diagnosis serum group onset and die, the botulinum toxin
contained in the sample is determined as this type of botulinum toxin.
Note. If the sample supernatant without being subjected to trypsin activation
treatment shows positive in the toxin detection test or confirmation test, the
trypsin activation treatment test may be omitted from the toxicity test or type
test.
5.3 Clostridium botulinum test
5.3.1 Enrichment culture and detection test
5.3.1.1 TAKE 4 pieces of meat broth and 2 TPGY broth tubes; MAKE it boil in
oxygen; COOL it rapidly; DO not shake it; slowly ADD trypsin solution into the
broth below the liquid paraffin level in the TPGY broth tube, 1 mL for each tube;
PREPARE it into TPGYT.
5.3.1.2 PIPETTE 2 mL of the sample homogenate or the centrifuge sediment
suspension produced in the toxin preparation process; INOCULATE it into the
meat culture medium, 4 pieces for each set of sample; MAKE 2 pieces directly
subjected to anaerobic culture at 35 °C ± 1 °C for 5 d; PLACE the other two
pieces at 80 °C for 10 min and then MAKE them subjected to anaerobic culture
at 35 °C ± 1 °C for 5 d; USE the same method to inoculate another 2 TPGYT
Note. During inoculation, USE a sterile pipette to gently draw the sample
homogenate or centrifuge sediment suspension; carefully INSERT the pipette
tip into the bottom of the broth tube; slowly ADD the sample solution into the
broth; DO not stir it or blow air.
1 °C for 48 h; FOLLOW the requirements of 5.3.2.3 to observe the colony
morphology and its purity.
5.3.3 Identification test
5.3.3.1 Stain microscopy
PICK the suspicious colonies for smearing, Gram staining and microscopy;
in oval, larger than the bacteria, and located in the secondary end, AND the
bacteria is tennis racket-like.
5.3.3.2 Toxin gene detection
a) Strain activation. PICK the suspicious colonies or the strains to be
identified; INOCULATE it onto the TPGY; MAKE it subject to anaerobic
culture at 35 °C ± 1 °C for 24 h
b) DNA template preparation. PIPETTE 1.4 mL of TPGY culture medium
into a sterile centrifuge tube; MAKE it subject to 14000 × g centrifugation
for 2 min; DISCARD the supernatant; ADD 1.0 mL of PBS suspension
the supernatant; USE 400 μL of PBS for resuspending the sediment;
ADD 100 µL of 10 mg/mL lysozyme solution; SHAKE it uniformly; PLACE
it in 37 °C water bath for 15 min; ADD 10 µL of 10 mg/mL protease K
solution; SHAKE it uniformly; PLACE it in 60 °C water bath for 1 h; MAKE
it subject to boiling water bath for another 10 min; MAKE it subject to
14000 × g centrifugation for 2 min; TRANSFER the supernatant into a
sterile centrifuge tube; ADD 50 µL of 3 mol/L NaAc solution and 1.0 mL
of 95% ethanol; SHAKE it uniformly; PLACE it at -70 °C or -20 °C for 30
min; MAKE it subject to 14000 × g centrifugation for 10 min; DISCARD
buffer solution; PRESERVE it ...
|