Foreword
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This standard replaces GB 4789.35-2016 National food safety standard - Food microbiological examination - Examination of lactic acid bacteria.
The following main changes have been made with respect to GB 4789.35-2016:
——the real-time PCR method has been added as an optional method;
——the definition, sample preparation and culture time of lactic acid bacteria have been modified;
——the description of counting method of S.thermophilus and Lactobacillus has been modified; and
——the composition of some media, the concentration of stock solution and the preparation method have been modified.
National food safety standard -
Food microbiological examination - Examination of lactic acid bacteria
1 Scope
This standard specifies the examination method of lactic acid bacteria in foods containing lactic acid bacteria.
This standard is applicable to the examination of lactic acid bacteria in foods containing active lactic acid bacteria.
2 Terms and definitions
2.1 lactic acid bacteria
a generic term for bacteria that can mainly produce a large amount of lactic acid by fermenting sugar, cannot liquefy gelatin, will not produce indole, are Gram-positive, have no motility, have no spores, and have negative reaction with catalase, nitrate reductase and cytochrome oxidase. The lactic acid bacteria in this standard mainly include Lactobacillus, Bifidobacterium and Streptococcus thermophilus.
3 Equipment and materials
In addition to the conventional sterilization and culture equipment in microbiological laboratory, other equipment and materials are as follows.
3.1 Thermostatic incubator: 36℃±1℃.
3.2 Anaerobic culture device: anaerobic incubator, anaerobic jar, anaerobic bag or device that can provide the same anaerobic effect.
3.3 Refrigerator: 2℃-8℃.
3.4 Homogenizer and aseptic homogenizing bag, homogenizing cup or sterilized mortar.
3.5 Vortex mixer.
3.6 Electronic balance: with a sensitivity of 0.001g.
3.7 Real-time quantitative PCR instrument.
3.8 Thermostatic water bath or metal bath.
3.9 Centrifuge: with a centrifugal force of greater than 10,000×g.
3.10 Aseptic test tubes: 18mm×180mm and 15mm×100mm.
3.11 Aseptic pipettes: 1mL (with a scale division of 0.01mL) and 10mL (with a scale division of 0.1mL).
3.12 Micropipettors and sterile tips: 2μL, 10μL, 100μL, 200μL and 1,000μL.
3.13 Aseptic conical flasks: 500mL and 250mL.
3.14 Aseptic plate: with a diameter of 90mm.
3.15 PCR tube.
4 Media and reagents
4.1 Diluent: see A.1 in Annex A.
4.2 MRS (Man Rogosa Sharpe) agar medium: see A.2 in Annex A.
4.3 Li-mupirocin and cysteine hydrochloride modified MRS agar medium: see A.3 in Annex A.
4.4 MC (Modified Chalmers) agar medium: see A.4 in Annex A.
4.5 0.5% sucrose fermentation tube: see A.5 in Annex A.
4.6 0.5% cellobiose fermentation tube: see A.5 in Annex A.
4.7 0.5% maltose fermentation tube: see A.5 in Annex A.
4.8 0.5% mannitol fermentation tube: see A.5 in Annex A.
4.9 0.5% salicin fermentation tube: see A.5 in Annex A.
4.10 0.5% sorbitol fermentation tube: see A.5 in Annex A.
4.11 0.5% lactose fermentation tube: see A.5 in Annex A.
4.12 Aesculin fermentation tube: see A.6 in Annex A.
4.13 Gram staining solution: see A.7 in Annex A.
4.14 Normal saline: see A.8 in Annex A.
4.15 DNA extracting solution: see A.9 in Annex A.
4.16 10×PCR buffer solution: see A.10 in Annex A.
4.17 Li-mupirocin (C26H43O9·Li): chemically pure.
4.18 Cysteine hydrochloride (C3H8ClNO2S): with a purity of greater than 99%.
4.19 dNTPs.
4.20 Taq DNA polymerase: 5U/μL.
4.21 Seven kinds of lactic acid bacteria primers and probes.
4.22 Sterilized deionized water.
Foreword i
1 Scope
2 Terms and definitions
3 Equipment and materials
4 Media and reagents
5 Examination procedure
6 Operation steps
7 Results and report
8 Identification of lactic acid bacteria (optional)
Annex A Media and reagents
