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GB 4789.12-2025: National food safety standard - Food microbiological examination - Examination of Clostridium botulinum toxin and botulinum toxin
Status: Valid GB 4789.12: Evolution and historical versions
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| GB 4789.12-2025 | English | 299 |
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National food safety standard - Food microbiological examination - Examination of Clostridium botulinum toxin and botulinum toxin
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GB 4789.12-2025
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| GB 4789.12-2016 | English | 85 |
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National food safety standard - Food microbiological examination - Clostridium botulinum and botulinum toxin test
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GB 4789.12-2016
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| GB/T 4789.12-2003 | English | 199 |
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Microbiological examination of food hygiene -- Examination of Clostridium botulinum and botulinus toxin
| Obsolete |
GB/T 4789.12-2003
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| GB 4789.12-1994 | English | RFQ |
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Microbiological examination of food hygiene. Examination of Clostridium botulinum and botulinus toxin
| Obsolete |
GB 4789.12-1994
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| GB 4789.12-1984 | English | RFQ |
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Microbiological examination of food hygiene--Examination of Clostridium botulinum and botulinus toxin
| Obsolete |
GB 4789.12-1984
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Basic data | Standard ID | GB 4789.12-2025 (GB4789.12-2025) | | Description (Translated English) | National food safety standard - Food microbiological examination - Examination of Clostridium botulinum toxin and botulinum toxin | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Word Count Estimation | 15,111 | | Date of Issue | 2025-09-02 | | Issuing agency(ies) | National Health Commission; State Administration for Market Regulation | GB 4789.12-2025: National food safety standard - Food microbiological examination - Examination of Clostridium botulinum toxin and botulinum toxin
---This is an excerpt. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.), auto-downloaded/delivered in 9 seconds, can be purchased online: https://www.ChineseStandard.net/PDF.aspx/GB4789.12-2025
National Standards of the People's Republic of China
National Food Safety Standards
Food microbiology testing
Clostridium botulinum and botulinum toxin testing
Published on 2025-09-02
Implemented on 2026-03-02
National Health Commission of the People's Republic of China
State Administration for Market Regulation issued
Foreword
This standard replaces GB 4789.12-2016 "National Food Safety Standard - Microbiological Examination of Food - Clostridium botulinum and Botulinum Toxin Detection".
Test.
Compared with GB 4789.12-2016, the main changes in this standard are as follows.
---Added terms and definitions;
---Added a real-time fluorescent PCR identification method for Clostridium;
---The standard name has been modified;
---The range has been modified;
---Modifications were made to the equipment and materials, culture media and reagents;
---The testing procedures, operating steps, results and reports, and appendices have been revised.
National Food Safety Standards
Food microbiology testing
Clostridium botulinum and botulinum toxin testing
1.Scope
This standard applies to the testing of Clostridium botulinum and botulinum toxin in food.
2.Terms and Definitions
Clostridium botulinum
Gram-positive anaerobic bacilli that can form oval-shaped spores. These spores are generally larger than the bacterial cell and located at the subterminal end. Under suitable culture conditions, they can produce...
tyricum), etc.
3.Equipment and Materials
In addition to the routine sterilization and culture equipment for the microbiology laboratory, the other equipment and materials are as follows.
3.1 Refrigerator. 2℃~8℃, -18℃~-20℃, -80℃.
3.2 Balance. Sensitivity 0.1g, 0.001g.
3.3 pH meter, pH colorimetric tubes or precision pH test paper.
3.4 Sterile scissors, tweezers, and reagent spoons.
3.5 Homogenizer or sterile mortar.
3.6 Centrifuges. 3000g, 14000g.
3.7 Anaerobic culture apparatus. anaerobic incubator, anaerobic tank, anaerobic bag or apparatus that provides equivalent anaerobic effect.
3.8 Constant temperature incubator. 36℃±1℃, 28℃±1℃.
3.9 Temperature control devices. 36℃±1℃, 60℃±1℃, 80℃±1℃, 100℃±1℃.
3.10 Microscope. 100×~1000×.
3.11 Conventional PCR instrument and real-time fluorescence PCR instrument.
3.12 Ordinary electrophoresis apparatus or capillary electrophoresis apparatus.
3.13 Gel imaging system or ultraviolet detector.
3.14 Nucleic acid protein analyzer or ultraviolet spectrophotometer.
3.15 Micropipettes and matching sterile pipette tips. 2μL, 10μL, 20μL, 100μL,.200μL, 1000μL.
3.16 Sterile pipettes. 1 mL (with 0.01 mL graduations), 10 mL (with 0.1 mL graduations), 25 mL (with 0.1 mL graduations).
3.17 Sterile conical flask. 100mL.
3.18 Petri dish. 90mm in diameter.
3.19 Centrifuge tubes. 1.5mL, 5mL, 50mL.
3.20 Sterile filter. Filter membrane pore size 0.45μm, 0.22μm.
3.21 PCR reaction tube.
3.22 Sterile syringe. 1 mL.
3.23 Mice. weighing 15g~20g, each batch of experiments should use KM (Kunming) or ICR mice of the same strain and single sex.
The weight difference between individual mice should not exceed ±20% of the average weight, and if the animal is female, it should not have mated or been pregnant.
4.Culture media and reagents
Unless otherwise specified, reagents used in PCR experiments shall be analytical grade or meet biochemical reagent standards, and the water used for preparing culture media shall meet the following requirements.
The requirements in GB 4789.28.
4.1 Physiological saline. see A.1.
4.2 Meat culture medium. see A.2.
4.3 Trypsin-peptone-glucose-yeast extract broth (TPGYT) medium. see A.3.
4.4 Egg yolk agar medium. see A.4.
4.5 Columbia blood agar medium. see A.5.
4.6 Gelatin phosphate buffer. see A.6.
4.7 Phosphate-buffered saline (PBS). see A.7.
4.8 Gram staining solution. see A.8.
4.9 Trypsin. activity 1.250 or 0.25%.
4.10 Botulinum toxin diagnostic serum. mixed type, type A, type B, type E, type F.
4.11 1 mol/L sodium hydroxide.
4.12 1mol/L hydrochloric acid.
4.13 Anhydrous ethanol and 95% ethanol.
4.14 10 mg/mL lysozyme solution.
4.15 10 mg/mL proteinase K solution.
4.16 3 mol/L sodium acetate solution (pH 5.2).
4.17 TE buffer.
4.18 Primers and probes. Prepare them to a concentration of 10 μmol/L using sterile ultrapure water or TE buffer immediately before use.
4.19 10×PCR buffer. 0.5 mmol/L KCl, 0.1 mmol/L Tris-HCl (pH 8.3), 0.015 mmol/L MgCl2.
4.20 25 mmol/L MgCl2.
4.21 dNTPs. The concentrations of dATP, dTTP, dCTP, and dGTP were all 10 mmol/L.
4.22 Taq DNA polymerase. 5 U/μL.
4.23 Agarose. Electrophoresis grade.
4.24 Ethidium bromide, Goldview or other nucleic acid dyes.
4.25 5×TBE or 10×TAE buffer.
4.26 6× loading buffer.
4.27 DNA Molecular Quality Standards. Covering target band length.
5.Inspection Procedure
The testing procedure for Clostridium botulinum and botulinum toxin is shown in Figure 1.
Note. Report (I). No botulinum toxin (a certain type) was detected in the sample; Report (II). Clostridium botulinum (a certain type) was detected in the sample;
Report (III). No Clostridium botulinum producing (a certain type) of botulinum toxin was detected in the sample.
Figure 1.Procedure for detecting Clostridium botulinum and botulinum toxin.
6.Operating Steps
6.1 Sample Preparation
For solid, semi-solid, and difficult-to-absorb liquid foods, weigh 25g aseptically and place them in a sterile homogenizing bag or sterile mortar. For block foods, use...
Chop aseptically. For foods with high water content, add 25 mL of gelatin phosphate buffer; for foods with low water content, such as milk powder and beef jerky, add...
Add 50 mL (or more; the volume of the diluent can be adjusted according to the moisture content of the sample, generally diluted to a level suitable for inoculation) of gelatin phosphate buffer.
Soak the sample in the solution for 30 minutes, then beat it with a tapping homogenizer for 1 to 2 minutes or grind it with a sterile pestle to prepare a homogenized solution, and collect it for later use.
For easily absorbed liquid foods, shake well and take 25mL for later use.
Note. Remaining samples after sampling should be refrigerated at 2℃~8℃ until the test report is issued. Samples that test positive will be sterilized by pressure steam sterilization.
For harmless treatment, it is recommended to sterilize by high-pressure steam at 121℃ for more than 30 minutes.
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