GB 4789.12-2025 English PDFGB 4789.12: Historical versions
Basic dataStandard 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 4789.12-2016: National food safety standard - Food microbiological examination - Clostridium botulinum and botulinum toxin test---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order. National Food Safety Standard - Botulinum Toxin Clostridium Botulinum and - Food Microbiological Examination National Standards of People's Republic of China National Food Safety Standard Microbiological examination of food Clostridium botulinum and inspection Published 2016-12-23 2017-06-23 implementation National Health and Family Planning Commission People's Republic of China China Food and Drug Administration issued ForewordThis standard replaces GB/T 4789.12-2003 "Microbiological examination of food hygiene Clostridium botulinum and inspection." As compared with the present standard GB/T 4789.12-2003, major changes are as follows. --- standard name was changed to "national food safety standards of food microbiological examination of Clostridium botulinum and inspection"; --- increased PCR identification method; --- increased results and reporting; --- Added Appendix A; --- modified equipment and materials; --- modify the media and reagents; --- revised inspection procedures; --- standardized sample preparation; --- modify the enrichment procedure and the test methods section isolated and cultured. National Food Safety Standard Microbiological examination of food Clostridium botulinum and inspection1 ScopeThis standard specifies the test Clostridium botulinum in foods (Clostridiumbotulinum) and botulinum toxin (botulinumtoxin) of method. This standard applies to food inspection in Clostridium botulinum and botulinum toxin.2 Equipment and MaterialsIn addition to the conventional sterilization and microbiological laboratory culture equipment, other equipment and materials as follows. Refrigerator 2.1. 2 ℃ ~ 5 ℃, -20 ℃. 2.2 Balance. a sense of the amount of 0.1g. 2.3 sterile surgical scissors, forceps, spoon reagent. 2.4 homogenizer or sterile mortar. 2.5 Centrifuge. 3000r/min, 14000r/min. 2.6 anaerobic apparatus. 2.7 Incubator. 35 ℃ ± 1 ℃, 28 ℃ ± 1 ℃. 2.8 constant temperature water bath. 37 ℃ ± 1 ℃, 60 ℃ ± 1 ℃, 80 ℃ ± 1 ℃. Microscope 2.9. 10 to 100 times. 3.6 10% trypsin solution. see A.6. 3.7 phosphate buffered saline (PBS). see A.7. 3.8 1mol/L sodium hydroxide solution. 3.9 1mol/L hydrochloric acid solution. 3.10 botulinum toxin serum diagnosis. 3.11 absolute ethanol and 95% ethanol. 3.12 10mg/mL lysozyme solution. 3.13 10mg/mL proteinase K solution. 3.14 3mol/L sodium acetate solution (pH5.2). 3.15 TE buffer. 3.16 primer. Table 1 The sequences of the synthetic, with ultrapure water for the extemporaneous preparation of primer concentration 10μmol/L. 3.17 10 × PCR buffer. 3.18 25mmol/LMgCl2. 3.19 dNTPs. dATP, dTTP, dCTP, dGTP. 3.20 Taq enzyme. 3.21 agarose. electrophoresis grade. 3.22 ethidium bromide or Goldview. 3.23 5 × TBE buffer. 3.24 6 × loading buffer. 3.25 DNA molecular weight standards.4 inspection proceduresClostridium botulinum and inspection procedures shown in Figure 1. Figure 1 Clostridium botulinum and botulinum toxin testing procedures Procedure 5 5.1 Sample Preparation 5.1.1 sample storage Samples to be tested should be placed 2 ℃ ~ 5 ℃ refrigerator. 5.1.2 solid and semi-solid food Solid or semi-solid little free liquid food sample is weighed aseptically 25g, placed in a sterile bag or sterile homogeneous mortar, block Aseptically minced food, solid foods higher moisture content of gelatin was added 25mL phosphate buffered saline, milk, beef jerky low water content Food was added 50mL of gelatin phosphate buffer, soaking 30min, 2min or tapping with a sterile pestle homogenizer manufactured by slap Preparation of liquid sample uniform, collecting spare. 5.1.3 Liquid Food Shake the liquid food, aseptic amount of 25mL test. The remaining sample processing 5.1.4 The remaining sample was placed 2 ℃ ~ 5 ℃ refrigerator after sampling, the report issued until the test results, according to the requirements of non-infectious waste Injury treatment, samples should be detected positive pressure steam sterilization treatment and disposal embodiment. 5.2 botulinum toxin detection 5.2.1 Preparation of liquid toxins A sample was homogenized about 40mL or 25mL homogeneous liquid sample into a centrifuge tube, 3000r/min centrifugal 10min ~ 20min, collected The supernatant was placed in a sterile test tube is divided into two, one for the direct detection of endotoxin, endotoxin for detecting a trypsin treatment. Liquid-like The precipitate and liquid bottom product retains about 12 mL, resuspended, the suspension prepared spare precipitate. Trypsin treatment. The supernatant was adjusted to pH 6.2 with 1mol/L sodium hydroxide or 1mol/L hydrochloric acid, 9 parts of supernatant was added by 1 part 10% Trypsin (activity 1.250) solution, mixed and incubated for 60min 37 ℃, the reaction solution during gentle shaking occasionally. 5.2.2 detection test 5, extracted with a syringe needle were trypsinized and the supernatant was centrifuged supernatant mice were injected intraperitoneally 3, 0.5 mL each, and observed Record poisoning in the mouse 48h. Typical botulinum toxin poisoning symptoms appear within 24h, morbidity and mortality usually within 6h, its Mainly for the vertical hair, limbs limp, difficulty breathing, showing bellows breathing, waist and abdomen depression, like Feng Yao, usually caused by respiratory failure and death, Preliminary determination can be caused by botulinum toxin. If the mouse morbidity or mortality after 24h, the mice should be carefully observed symptoms, if necessary, the supernatant was concentrated Repeat the test to exclude botulinum toxin poisoning. If mice sudden death (within for 30 min) results in obvious symptoms, should toxin into the supernatant OK appropriate dilution, the test is repeated. Note. toxins in animal testing should follow the provisions of GB "national food safety standards for food toxicology laboratory practices" of 15193.2. 5.2.3 confirmatory test Supernatant or (and) trypsinized toxin test positive supernatants, taken 3 parts of the respective test solution, 0.5 mL each, wherein the first plus Multi botulinum toxin type mixing equal amounts of serum diagnosis, mixed, incubated for 37 [deg.] C for 30 min; second equal amount of gelatin phosphate buffer, boiled after mixing Boiling 10min; third parts with the same amount of gelatin phosphate buffer, and mix. Triplicate each mixture were intraperitoneally injected two mice, each 0.5mL, observe and death within 96h poisoned mice. Results found. if first and second parts of the injection mixture of untreated mice died, while the third mixture mice incidence of death, and the meat appears The specific symptoms of toxic poisoning, it is determined that botulinum toxin was detected in the test sample. 5.2.4 Bioassay (selected to do the project) Take confirmatory test positive test solution, diluted with gelatin phosphate buffer prepared certain multiple dilutions, such as 10-fold, 50-fold, 100-fold, 500-fold, etc., each of the two intraperitoneal injection of mice, each 0.5 mL, the mice were observed, and morbidity and mortality recorded 96h to the case, the lowest calculated Lethal dose (MLD/mL or MLD/g), samples were evaluated virulence toxin botulinum, MLD is equal to all the mice died highest dilution Multiplied by the sample dilution test solution. For example, a sample of the supernatant was diluted twice prepared, and diluted 100-fold test mice were all died, And 500-fold dilution group survived, the virulence of the sample 200MLD/g. 5.2.5 Approval Test (selected to do the project) The toxicity measurement result, with gelatin phosphate buffer and the supernatant was diluted to 10MLD/mL ~ 1000MLD/mL as specified Type test solution, an equal amount of serum was mixed with each of the single botulinum toxin diagnosis (serum diagnosis is generally made of a lyophilized serum with physiological saline 1mL Dissolved in water), incubated for 37 ℃ 30min, two mice were injected intraperitoneally, each 0.5 mL, the mice were observed and recorded to the morbidity and mortality 96h. Meanwhile, instead of the diagnostic serum with gelatin phosphate buffer, mixed with equal amounts of test solution as a test control mice. Results determined. type of a single diagnostic serum and normal animals survived disease-free, while the control group and the other animals single type of diagnosis serogroup incidence Death, it is determined that botulinum toxin type A botulinum toxin contained in the sample. Note. The supernatant of the samples treated without trypsin activation toxin detection test or confirmatory test is positive, the determination of the virulence and trypsin may be omitted laser type testing Live handling test. 5.3 Clostridium botulinum test 5.3.1 Test enrichment culture and detection 5.3.1.1 Remove the cooked meat medium 4 and the tube 2 TPGY broth, boiled impermeable 10min ~ 15min, dissolved oxygen excluded, the rapid cooling However, do not shake, in TPGY broth with trypsin solution was slowly added to the broth at the level of the liquid paraffin, each 1mL, prepared TPGYT. 5.3.1.2 draw fluid or homogenized samples were prepared by centrifugation of toxins during precipitation suspension inoculated into 2mL of cooked meat medium, then each sample 4 species, two placed directly 35 ℃ ± 1 ℃ anaerobic culture to 5D, the other two discharge 80 ℃ incubated 10min, then placed in 35 ℃ ± 1 ℃ anaerobic Grown to 5D; TPGYT same inoculation broth with 2, 28 ℃ ± 1 ℃ anaerobic culture to 5d. Note. the time of inoculation, with a sterile pipette solution or homogenized samples were centrifuged gently draw precipitation suspension, carefully insert the bottom of the suction pipe broth tubes, sample solution discharged slowly until the meat Soup, do not stir or blowing. 5.3.1.3 inspection records enrichment culture turbidity, gas production, meat residue particles digestive conditions, and note the smell. Clostridium botulinum culture is producing gas, Turbid broth (cooked meat medium Clostridium botulinum type A and type B black broth), digestion or indigestion Rouli, odor was identified. 5.3.1.4 enrichment culture was taken for Gram staining, observation of cell morphology, note whether there spores, the relative proportions of spores, spores in Location within the cell. 5.3.1.5 If the enrichment culture was grown sterile 5d, 1Od should be extended to the culture, the growth was observed. 5.3.1.6 take positive enrichment culture supernatant tube, and a confirmatory test for toxin detection was conducted for the test method when necessary to 5.2, The positive results demonstrate the presence of Clostridium botulinum in the sample. Note. toxin test TPGYT enrichment broth without adding trypsin treatment. 5.3.2 Isolation and purification of culture 5.3.2.1 enrichment broth pretreatment, 1mL suction enrichment broth to a sterile screw cap tube, an equal volume of filter-sterilized ethanol, mixed Uniform, placed at room temperature for 1h. 5.3.2.2 take enrichment and enrichment broth cultures were treated with ethanol to egg yolk agar plate streaked, 35 ℃ ± 1 ℃ anaerobic culture 48h. 5.3.2.3 colony morphology was observed plate culture, colonies of C. botulinum flat or raised, smooth or rough, easily spread into growth, irregular edges The formed precipitate a cream-colored halo around the colonies (E wide type, A-type and B-type narrower), observed under oblique light, pearl-like surface of the colony presented Iridescent, this gloss region may be spread with the growth of irregular edges to the outer diffusion region halo. 5.3.2.4 pure culture strain, select the five Clostridium botulinum suspected isolated colonies on culture plates, were inoculated egg yolk agar plates, 35 ℃ ± 1 ℃, anaerobic culture 48h, 5.3.2.3 observed by colony morphology and purity. 5.3.3 identification test 5.3.3.1 staining Suspected colonies were picked smear, Gram staining and microscopic examination, Clostridium botulinum bacteria form thick Gram-positive bacteria, spores oval Shape, larger than the cells located in the end times, as a tennis racket-like cells. 5.3.3.2 toxin gene detection a) Activation Strain. suspected colonies were picked to be identified or strain is inoculated TPGY, 35 ℃ ± 1 ℃ anaerobic culture 24h. b) Preparation of DNA template. lessons TPGY broth to a sterile 1.4mL microcentrifuge tube, 14000 × g centrifugation 2min, the supernatant was discarded, 1.0mLPBS cell suspension was added, 14000 × g centrifugation 2min, the supernatant discarded, the precipitate was resuspended with 400μLPBS added 10mg/mL lysozyme solution 100μL, shake, 37 ℃ water bath for 15min, was added 10mg/mL proteinase K solution 10μL, shake, 60 ℃ water bath for 1h, and then boiling water bath for 10min, 14000 × g centrifugation 2min, the supernatant was transferred to a sterile little away Centrifuge tubes, was added 3mol/LNaAc 50μL solution and 1.0 mL of 95% ethanol, shake, or placed -70 ℃ -20 ℃ 30min, 14000 × g centrifugation 10min, the supernatant was discarded, the precipitate after drying was dissolved 200μLTE buffer, placed Paul -20 ℃ Memory backup. Note. The actual laboratory situation, conventional water may be used to prepare DNA template boil method or a commercial kit. c) determining the concentration of the nucleic acid (if necessary). Take 5μLDNA template solution, adding ultrapure water was diluted to 1mL, using a nucleic acid or protein analyzer UV spectrophotometer absorbance A260 and A280, respectively 260nm and 280nm band detection. According to formula (1) is calculated DNA concentration. When the concentration of 0.34μg/mL ~ 340μg/mL or A260/A280 ratio is between 1.7 to 1.9, suitable for PCR Amplification. C = A260 × N × 50 (1) Where. C --- DNA concentration, in micrograms per milliliter (μg/mL); Absorbance at A260 --- 260nm; N --- nucleic acid dilution. d) PCR amplification. 1) were used for PCR amplification for each type of botulinum toxin genes designed specific primers (see Table 1), comprising A Botulinum toxin (botulinumneurotoxinA, bont/A), B botulinum toxin (botulinumneurotoxinB, bont/B), E botulinum toxin (botulinumneurotoxinE, bont/E) and F botulinum toxin (botulinum neurotoxinF, bont/F), each PCR reaction tube to detect one other Clostridium botulinum type. Table 1 Primer sequences and Clostridium botulinum PCR product was detected toxin gene Clostridium botulinum type detection primer sequence length of the amplification/bp A type F5'-GTGATACAACCAGATGGTAGTTATAG-3 ' R5'-AAAAAACAAGTCCCAATTATTAACTTT-3 ' Type B F5'-GAGATGTTTGTGAATATTATGATCCAG-3 ' R5'-GTTCATGCATTAATATCAAGGCTGG-3 ' E-type F5'-CCAGGCGGTTGTCAAGAATTTTAT-3 ' R5'-TCAAATAAATCAGGCTCTGCTCCC-3 ' Type F F5'-GCTTCATTAAAGAACGGAAGCAGTGCT-3 ' R5'-GTGGCGCCTTTGTACCTTTTCTAGG-3 ' 2) formulation of the reaction system are shown in Table 2, the amount of each reagent in the reaction system may be different depending on the circumstances or the total volume of reaction Adjusted accordingly. Table 2 PCR reaction was detected Clostridium botulinum toxin genes Volume of reagent added to a final concentration of/μL 10 × PCR buffer, 1 × 5.0 25mmol/LMgCl2 2.5mmol/L 5.0 10mmol/LdNTPs 0.2mmol/L 1.0 10μmol/L forward primer 0.5μmol/L 2.5 10μmol/L reverse primer 0.5μmol/L 2.5 5U/μLTaq enzyme 0.05U/μL 0.5 DNA template - 1.0 ddH2O - 32.5 Total Volume - 50.0 3) reaction sequence, denaturation 95 ℃, 5min; cycle parameters 94 ℃, 1min, 60 ℃, 1min, 72 ℃, 1min; number of cycles 40; rear extension 72 ℃, 10min; 4 ℃ for use. 4) PCR amplification system should be set to a positive control, a negative control and blank control. Clostridium botulinum strains containing known or containing meat Quality control materials poison toxin gene as a positive control, non-Clostridium botulinum genomic DNA as a negative control, sterile water as a blank Control. e) gel electrophoresis PCR products formulated from 1.2% to 1.5% agarose gel with 0.5 × TBE buffer gel was heated After thawing ethidium bromide was added to 0.5μg/mL or Goldview5μL/100mL prepared bun was cooled to about 60 ℃, taking Amplification products were mixed with 10μLPCR 2.0μL6 × loading buffer, spotted, wherein a well of DNA molecular weight standards. 0.5 × TBE electrophoresis buffer, 10V/cm constant voltage electrophoresis, electrophoresis time is determined in accordance with the movement position of bromophenol blue, UV detection Tester or gel imaging system to observe and record the results. PCR amplification products may also be detected by capillary electrophoresis apparatus. f) determining a result, the negative control and blank control strips were not there, the positive control amplified bands appeared expected size (see Table 1), The PCR assay was established is determined; sample to be tested in amplification bands of the expected size, it is determined that PCR positive results, according to Table 1 Determining strains of Clostridium botulinum types, test samples were not expected size of amplified bands appeared, the determination result is negative for PCR. NOTE. PCR test environment condition and process control "control specification molecular biology laboratory quality food" shall refer to provisions GB/T 27403. 5.3.3.3 Test toxigenic strain The PCR positive or suspected strains of Clostridium botulinum strain was inoculated TPGYT broth or cooked meat medium (for C. botulinum type E), press 5.3.1.2 5D anaerobic culture conditions, for toxin detection and (or) approval test, the toxin test positive by 5.2 confirmatory method, is determined meat Clostridium toxin, Clostridium botulinum typed determined according to finalization of the test results. NOTE. The PCR-positive strains typed, the serum can be diagnosed directly with another botulinum toxin type appropriate confirmatory tests.6 results report6.1 botulinum toxin detection results report 5.2.2 and 5.2.3 The test results, reported 25g detected or not detected botulinum toxin (mL) sample. 5.2.5 The approval test results reported 25g (mL) samples of a botulinum toxin detected. 6.2 Clostridium botulinum test results report 5.3 According to the test results, report detected or not detected in the sample or the detection of Clostridium botulinum Clostridium botulinum certain type.Appendix AMedia and Reagents A.1 cooked meat medium A.1.1 ingredients Fresh beef 500.0g Peptone 30.0g Yeast extract 5.0g 5.0g of sodium dihydrogen phosphate Glucose 3.0g Soluble starch 2.0g Distilled water 1000.0mL A.1.2 Method 500.0g was weighed fresh beef fat and fascia was removed and chopped, and 1000mL of distilled water was added 1mol/L sodium hydroxide solution 25mL, boiled with stirring 15min, cooled sufficiently, the surface fat was removed, filtered through gauze and extruded meat residue raffinate, and meat broth were collected slag. Broth was added in the ingredient list and other substances made up with distilled water to 1000mL, adjusted to pH 7.4 ± 0.1, semi-dry meat residue to cool. In 20mm × 150mm test tube was added to the ground meat residue 1cm ~ 2cm high, 0.1g ~ 0.2g added to each tube or a little reduced iron powder Iron, prepared broth was added 15mL, liquid paraffin overlay medium was added and finally 0.3cm ~ 0.4cm, 121 ℃ autoclaved 20min. A.2 trypsin tryptone glucose yeast extract broth (TPGYT) A.2.1 base component (TPGY broth) Trypticase (trypticase) 50.0g Peptone 5.0g Yeast extract 20.0g Glucose 4.0g 1.0g sodium thioglycollate Distilled water 1000.0...... |