GB 4789.12-2016_English: PDF (GB4789.12-2016)
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National Food Safety Standard -- Food Microbiological Examination -- Clostridium Botulinum and Botulinum Toxin
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GB 4789.12-2016
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Standards related to: GB 4789.12-2016
| Standard ID | GB 4789.12-2016 (GB4789.12-2016) | | Description (Translated English) | National Food Safety Standard -- Food Microbiological Examination -- Clostridium Botulinum and Botulinum Toxin | | Sector / Industry | National Standard | | Classification of Chinese Standard | X09 | | Word Count Estimation | 13,168 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB/T 4789.12-2003 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | GB 4789.12-2016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
SHAKE the liquid food uniformly; USE the sterile operation to MEASURE 25
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
TAKE about 40 mL of sample homogenate or 25 mL of homogenized liquid
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
37 °C for 60 min, during which gently SHAKE the reaction solution at certain
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
phosphate buffer solution to dilute the supernatant 10 MLD/mL ~ 1000
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
does not onset but survives normally, BUT the animals in the control group or
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
water (but separated from water) for 10 min ~ 15 min; REMOVE the dissolved
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
broth tubes; MAKE them subject to anaerobic culture at 28 °C ± 1 °C to 5 d.
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;
AND the clostridium botulinum cell morphology is Gram-positive bacilli, spores
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
bacteria; MAKE it subject to 14000 × g centrifugation for 2 min; DISCARD
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
the supernatant; DRY the sediment and DISSOLVE it into 200 µL of TE
buffer solution; PRESERVE it ...
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