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GB 4789.40-2024 PDF English

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GB 4789.40-2024: National food safety standard - Food Microbiological Testing - Cronobacter Testing
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GB 4789.40: Evolution and historical versions

Standard IDContents [version]USDSTEP2[PDF] deliveryName of Chinese StandardStatus
GB 4789.40-2024English260 Add to Cart 0-9 seconds. Auto-delivery National food safety standard - Food Microbiological Testing - Cronobacter Testing Valid
GB 4789.40-2016English70 Add to Cart 0-9 seconds. Auto-delivery National food safety standard - Food Microbiological Examination - Examination of Cronobacter (Enterobacter Sakazakii) Obsolete
GB 4789.40-2010English399 Add to Cart 3 days National food safety standard -- Food microbiological examination: Enterobacter sakazakii Obsolete
GB/T 4789.40-2008English439 Add to Cart 4 days Microbiological examination of food hygiene -- Examination of enterobacter sakazakii Obsolete

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GB 4789.40-2024: National food safety standard - Food Microbiological Testing - Cronobacter Testing


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GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard - Food Microbiological Examination - Examination of Cronobacter Issued on. FEBRUARY 8, 2024 Implemented on. AUGUST 8, 2024 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation.

Table of Contents

Foreword... 3 1 Scope... 4 2 Equipment and Materials... 4 3 Culture Media and Reagents... 5 Method I Qualitative Examination of Cronobacter... 6 4 Examination Procedures... 6 5 Operating Steps... 7 6 Result and Report... 11 Method II Quantitative Examination of Cronobacter... 11 7 Operating Steps... 11 8 Result and Report... 11 Appendix A Culture Media and Reagents... 12 Appendix B Gene Enrichment Target Reference Sequence of Cronobacter... 19 Appendix C Cronobacter Most Probable Number (MPN) Retrieval Table... 20

Foreword

This Standard serves as a replacement of GB 4789.40-2016 National Food Safety Standard - Food Microbiological Examination - Examination of Cronobacter. In comparison with GB 4789.40-2016, the main changes are as follows. ---The title of the Standard is modified; ---The scope of application of this Standard is expanded to supplementary foods for infants and young children; ---PCR identification method is added as optional content; ---The yellow pigment-producing and amygdalin items in picking yellow colonies and biochemical identification are deleted; ---The pH adjustment method for culture media and reagents is modified; ---The preheating, selective enrichment temperature and TSA plate incubation temperature and time are modified; ---The culture temperature of biochemical reactions and the preparation of amino acid culture medium are modified. National Food Safety Standard - Food Microbiological Examination - Examination of Cronobacter

1 Scope

This Standard specifies the examination method for Cronobacter spp. in food. This Standard is applicable to the examination of cronobacter in formula foods and supplementary foods for infants and young children, milk and dairy products and their raw materials.

2 Equipment and Materials

In addition to the routine sterilization and culture equipment of the microbiology laboratory, other equipment and materials are as follows. 2.4 Balance. with a division value of 0.1 g and 0.01 g. 2.5 Oscillator. 2.6 Sterile pipette. 1 mL (with a scale of 0.01 mL), 10 mL (with a scale of 0.1 mL) or micropipette and tip. 2.7 Sterile container. with a capacity of 100 mL, 200 mL and 2,000 mL. 2.8 Sterile petri dish. with a diameter of 90 mm. 2.9 pH meter or pH colorimetric tube or precision pH test paper. 2.10 Microbial biochemical identification system. 2.11 PCR instrument. 2.12 Centrifuge. with a rotation speed  12,000 r/min. 2.13 Gel imaging system or UV detector. 2.14 Agarose horizontal electrophoresis instrument or capillary electrophoresis instrument. 2.15 PCR reaction tube. 2.16 1.5 mL centrifuge tube. 2.17 10 L inoculation loop.

3 Culture Media and Reagents

3.1 Buffered peptone water, BPW. see A.1. 3.2 Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm. see A.2. 3.3 Cronobacter chromogenic medium. 3.4 Trypticase soy agar, TSA. see A.3. 3.5 Biochemical identification kit. 3.6 Oxidase reagent. see A.4. 3.7 L-lysine decarboxylase medium. see A.5. 3.8 L-ornithine decarboxylase medium. see A.6. 3.9 L-arginine dihydrolase medium. see A.7. 3.13 5 U/L thermostable DNA polymerase. 3.14 2.5 mmol/dNTPs. dATP, dTTP, dCTP, dGTP. 3.15 25 mmol/L MgCl2. 3.17 Cronobacter quality control strain. ATCC 29544 or equivalent strain provided by an organization with strain preservation qualifications. 3.18 Escherichia coli quality control strain. ATCC 25922 or equivalent strain provided by an organization with strain preservation qualifications. 3.19 DNA extraction reagent. bacterial genomic DNA extraction kit. 3.20 Commercial PCR reaction master mix. 3.21 Standard (high melting point) agarose. analytically pure. 3.22 Nucleic acid stains. 3.23 Molecular mass standard. 100 bp DNA ladder.

4 Examination Procedures

The examination procedures of cronobacter are shown in Figure 1.

5 Operating Steps

5.1 Pre-enrichment and Selective Enrichment 5.2 Separation 5.2.1 Gently mix the mLST-Vm broth culture, use 10 L inoculation loop to respectively take PCR Identification (optional) 5.3 PCR Identification (optional) The PCR test environmental conditions and process control shall be implemented with reference to the stipulations of GB/T 27403. 5.3.1 DNA template preparation The thermal lysis method can be adopted to prepare the template. Pick 2 ~ 3 suspicious cronobacter colonies from each TSA plate into 500 L of sterilized deionized water. 5.3.2 PCR enrichment 5.3.2.1 Primer The primer information for PCR identification is shown in Table 1. 5.3.2.2 PCR reaction system The composition of the PCR reaction system is shown in Table 2. 5.3.3 Control settings In each PCR identification, use the cronobacter standard strain DNA template as a positive control, and the Escherichia coli standard strain DNA template as a negative control. During the extraction process, set the sterilized deionized water as a DNA extraction blank control. The PCR reaction requires additional sterilized deionized water as a blank control for the PCR reaction system. 5.3.4 Electrophoresis 5.4 Confirmatory Test When 5.3 is selected, pick colonies from TSA plates with positive PCR result for biochemical identification. TSA plates with negative PCR result will no longer be subjected to the biochemical identification.

6 Result and Report

In accordance with the colony characteristics, confirmatory test (biochemical identification) and / or PCR identification results, report whether or not cronobacter was detected in the 100 g (mL) of sample.

7 Operating Steps

7.1 Sample Dilution 7.2 Separation and Identification

8 Result and Report

Based on the colony characteristics, confirmation test (biochemical identification) or PCR identification results, in accordance with the number of positive tubes, in which, cronobacter was detected, check the MPN retrieval table, and report the MPN value of cronobacter in 100 g (mL) of sample (see Table C.1 in Appendix C).

Appendix A

Culture Media and Reagents A.1 Buffered Peptone Water, BPW A.1.1 Ingredients Peptone 10.0 g Sodium chloride 5.0 g Disodium hydrogen phosphate 9.0 g Potassium dihydrogen phosphate 1.5 g Distilled water 1,000 mL A.2.2 Vancomycin solution A.2.2.1 Ingredients Vancomycin 10.0 mg Distilled water 10.0 mL A.2.3 Modified lauryl sulfate tryptose broth-vancomycin medium, mLST-Vm For each 10 mL of mLST, add 0.1 mL of vancomycin solution. The final concentration of vancomycin in the mixed solution is 10 g/mL. NOTE. mLST-Vm needs to be used within 24 hours. A.3 Trypticase Soy Agar, TSA A.3.1 Ingredients Tryptone 15.0 g Plant peptone 5.0 g Sodium chloride 5.0 g Agar 15.0 g Distilled water 1,000 mL A.4 Oxidase Reagent A.4.1 Ingredients N, N, N, N-tetramethyl-p-phenylenediamine hydrochloride 1.0 g Distilled water 100 mL A.4.2 Preparation method Freshly prepare a small amount. Store in the refrigerator away from light and use within 7 days. A.4.3 Test method Use a glass rod or disposable inoculation needle to pick a single characteristic colony and spread it on a filter paper plate moistened with oxidase reagent. If the filter paper does not turn fuchsia, purple or dark blue within 10 seconds, then, the oxidase test is negative, otherwise, the oxidase test is positive. NOTE. DO NOT USE nickel / chromium materials in the experiments. A.5 L-lysine decarboxylase medium A.5.1 Ingredients L-lysine monohydrochloride 5.0 g Yeast extract 3.0 g Glucose 1.0 g Bromocresol purple 0.015 g Distilled water 1,000 mL

Appendix B

Gene Enrichment Target Reference Sequence of Cronobacter The gene enrichment target reference sequence of cronobacter is as follows. NOTE. the bold part is the primer synthesis reference sequence.

Appendix C

Cronobacter Most Probable Number (MPN) Retrieval Table The retrieval table of most probable number (MPN) of cronobacter in every 100 g (mL) of test sample is shown in Table C.1. ......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.


      

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