GB 4789.12-2025National food safety standard - Food microbiological examination - Examination of Clostridium botulinum toxin and botulinum toxin (English PDF)

1 Scope

GB 4789.12-2025 is the Chinese national food safety standard for detecting botulinum toxin-producing clostridia and botulinum neurotoxin in food. Botulism is rare and catastrophic: the toxin is the most poisonous substance known, a few nanograms will kill, and in China the classic vehicles are home-fermented bean products, cured meats and improperly canned foods. Detection is unusual in that both the organism and its toxin must be sought, and the toxin assay determines the type as well as the presence. The standard sets the equipment and materials, the culture media and reagents, the testing procedure, the operating steps and the results and reporting, with an annex giving the media and reagent formulations. It takes effect on 2 March 2026.

This standard specifies the test methods for botulinum toxin-producing Clostridium and botulinum neurotoxin in food. This standard applies to the testing for botulinum toxin-producing Clostridium and botulinum neurotoxin in food.

2 Terms and definitions

botulinum toxin-producing Clostridium A collective term for a type of Gram-positive anaerobic bacilli that form oval-shaped spores, which are generally larger than a bacterial cell and located at the subterminal end, and are capable of producing botulinum neurotoxin under suitable culture conditions. This includes Clostridium botulinum and Clostridium butyricum, among others.

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 °C~8 °C, -18 °C~-20 °C, -80 °C.

3.2 Balance. The sensitivities are

0.1 g and

0.001 g.

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. 3000 g and 14000 g.

3.7 Anaerobic culture apparatus. anaerobic incubator, anaerobic tank, anaerobic bag or apparatus that provides equivalent anaerobic effect.

3.8 Constant temperature incubator. 36 °C±1 °C and 28 °C±1 °C.

3.9 Constant temperature devices. 36 °C±1 °C, 60 °C±1 °C, 80 °C±1 °C, and 100 °C±1 °C.

3.10 Microscope. 100×~1000×.

3.11 Conventional PCR instrument and real-time fluorescence PCR instrument.

4 Culture media and reagents

Unless otherwise specified, reagents used in PCR experiments shall be analytical grade or conform to biochemical reagent standards, and water used for culture medium preparation shall meet the requirements of GB 4789.28.

4.1 Physiological saline. see A.1.

4.2 Cooked meat culture medium. see A.2.

4.27 DNA Molecular Mass Standard. Covering target band length.

5 Testing procedure

The testing procedure for botulinum toxin-producing Clostridium and botulinum neurotoxin is shown in Figure 1.

6 Operating steps

6.1 Sample preparation For solid, semi-solid, and difficult-to-pipette liquid foods, weigh 25 g aseptically and place them in a sterile homogenizing bag or sterile mortar. For block-shaped foods, chop them 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 50 mL (or more; the volume of the diluent can be adjusted according to the moisture content of the sample, generally diluted to the point where it can be inoculated) of gelatin phosphate buffer. Soak for 30 minutes, then homogenize using a sterile homogenizer for 1 min~2 min or grind with a sterile pestle to prepare a homogenate. Collect the homogenate for later use. For easily pipetted liquid foods, shake well and take 25 mL for later use. NOTE. After sampling, the remaining samples shall be refrigerated at 2 °C~8 °C until the test report is issued. Samples that test positive shall be subjected to unharmful treatment by pressure steam sterilization. It is recommended to sterilize under high-pressure steam at 121 °C for more than 30 minutes.