GB 4789.35-2023 PDF English
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| Standard ID | Contents [version] | USD | STEP2 | [PDF] delivery | Name of Chinese Standard | Status |
| GB 4789.35-2023 | English | 230 |
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National food safety standard - Food microbiological examination - Lactic acid bacteria
| Valid |
| GB 4789.35-2016 | English | 85 |
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National food safety standard -- Microbiological examination of food -- Examination of lactic acid bacteria
| Obsolete |
| GB 4789.35-2010 | English | 70 |
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National food safety standard -- Food microbiological examination: Lactic acid bacteria
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| GB/T 4789.35-2008 | English | 399 |
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Microbiological examination of food hygiene -- Examination of lactic acid bacteria in foods
| Obsolete |
| GB/T 4789.35-2003 | English | 319 |
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Microbiological examination of food hygiene -- Examination of Lactic acid bacteria in yoghurt beverage
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GB 4789.35-2023: National food safety standard - Food microbiological examination - Lactic acid bacteria
---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/GB4789.35-2023
GB
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standards -- Food Microbiology
Testing -- Lactobacillus Testing
Issued on. SEPTEMBER 06, 2023
Implemented on. MARCH 06, 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 Terms and definitions... 4
3 Equipment and materials... 4
4 Culture medium and reagents... 5
5 Inspection procedures... 6
6 Operation steps... 7
7 Result and report... 9
8 Identification of lactic acid bacteria (optional)... 10
Annex A Culture medium and reagents... 13
Foreword
This Standard replaces GB 4789.35-2016 "National food safety standard --
Microbiological examination of food -- Examination of lactic acid bacteria".
Compared with GB 4789.35-2016, the main changes in this Standard are as follows.
- Add real-time fluorescence PCR method as an optional method;
- Modify the definition of lactic acid bacteria, sample preparation, and culture time;
- Modify the description of the enumeration method for streptococcus thermophilus
and lactobacillus;
- Modify some culture medium components, stock solution concentrations and
preparation methods.
National Food Safety Standards -- Food Microbiology
Testing -- Lactobacillus Testing
1 Scope
This Standard specifies the testing methods for lactic acid bacteria in foods containing
lactic acid bacteria.
This Standard is applicable to the inspection of lactic acid bacteria in foods containing
active lactic acid bacteria.
2 Terms and definitions
2.1 lactic acid bacteria
A general name for a group of bacteria that ferment sugars and mainly produce large
amounts of lactic acid. It is a bacterium that cannot liquefy gelatin, does not produce
indole, is Gram-positive, non-motile, non-spore-bearing, catalase-negative, nitrate
reductase-negative and cytochrome oxidase-negative.
The lactic acid bacteria in this Standard are mainly Lactobacillus, Bifidobacterium and
Streptococcus thermophilus.
3 Equipment and materials
In addition to the routine sterilization and culture equipment in the microbiology
laboratory, other equipment and materials are as follows.
3.1 Constant temperature incubator. 36℃±1℃.
3.2 Anaerobic culture device. anaerobic incubator, anaerobic tank, anaerobic bag or
device that can provide equivalent anaerobic effect.
3.3 Refrigerator. 2℃~8℃.
3.4 Homogenizer and sterile homogenization bag, homogenization cup or sterilized
mortar.
3.5 Vortex mixer.
3.6 Electronic balance. division is 0.001 g.
3.7 Real-time PCR machine.
3.8 Thermostatic water bath or metal bath.
3.9 Centrifuge. centrifugal force >10000×g.
3.10 Sterile test tube. 18 mm×180 mm, 15 mm×100 mm.
3.11 Sterile pipette. 1 mL (with 0.01 mL graduation), 10 mL (with 0.1 mL graduation).
3.12 Micropipette and sterilized tips. 2 μL, 10 μL, 100 μL, 200 μL, 1000 μL.
4 Culture medium 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 Esculin fermentation tube. see A.6 in Annex A.
4.13 Gram stain solution. see A.7 in Annex A.
4.14 Normal saline. see A.8 in Annex A.
5 Inspection procedures
The inspection procedures for lactic acid bacteria are shown in Figure 1.
6 Operation steps
6.1 Sample preparation
6.1.1 All sample preparation processes should follow sterile operating procedures.
6.1.2 The diluent should be fully preheated at 36℃±1℃ for 15 min~30 min before
testing.
6.1.3 Frozen samples can be thawed at 2℃~5℃ first. The time does not exceed 18 h.
It can also be defrosted at a temperature not exceeding 45°C. The time does not exceed
15 min.
6.1.4 Solid and semi-solid samples. Weigh 25 g of sample aseptically. Place in a sterile
homogenizing cup containing 225 mL of diluent. Homogenize at 8000 × g ~ 10000 × g
for 1 min ~ 2 min to prepare a 1.10 sample homogeneous solution. Or place in a sterile
homogenization bag with 225 mL of diluent. Use a slap homogenizer to beat for 1 min
~ 2 min to prepare a 1.10 sample homogeneous solution.
6.1.5 Liquid sample. The liquid sample should be shaken thoroughly first. Then use a
sterile straw to take 25 mL of the sample and put it into a sterile Erlenmeyer flask
containing 225 mL of diluent (an appropriate number of sterile glass beads are preset
in the bottle) or a homogeneous bag. Shake thoroughly or beat with a slap homogenizer
for 1 min ~ 2 min to prepare a 1.10 sample homogeneous solution.
6.1.6 Food samples containing lactic acid bacteria that have been processed by special
technologies (such as embedding technology) should be effectively pre-treated under
the corresponding technical/process requirements.
6.2 Dilution and culture
6.3 Lactic acid bacteria counting
6.3.1 Total number of lactic acid bacteria
The selection of culture conditions for counting the total number of lactic acid bacteria
and the explanation of the results are shown in Table 1.
6.3.2 Bifidobacteria counting
Based on the estimate of the bifidobacteria content of the sample to be tested, 2 to 3
consecutive appropriate dilutions are selected. Pipette 1 mL of sample homogeneous
solution for each dilution into a sterilized plate. Make two plates for each dilution. After
the dilution is transferred to the plate, pour 15 mL ~ 20 mL of mupirocin lithium salt
and cysteine hydrochloride modified MRS agar medium that have been cooled to
48℃~50℃ into the plate. Swirl the dish to mix evenly.
6.3.3 Streptococcus thermophilus counting
Based on the estimate of the viable number of streptococcus thermophilus in the sample
to be tested, 2 to 3 consecutive appropriate dilutions are selected. Pipette 1 mL of
sample homogeneous solution for each dilution into a sterilized plate. Make two plates
for each dilution. After the diluent is transferred to the plate, pour 15 mL ~ 20 mL of
MC agar medium cooled to 48℃~50℃ into the plate in time. Swirl the dish to mix
evenly. After the culture medium solidifies, place it upside down for aerobic culture at
36°C±1°C. According to the growth characteristics of streptococcus thermophilus,
culture is generally selected for 48 h. If the colony does not grow or grows small, you
can choose to culture it for 72 h. The colony characteristics of streptococcus
thermophilus on the MC agar medium plate are. the colonies are medium to small, red
colonies with neat and smooth edges, 2 mm±1 mm in diameter, and the back of the
colonies is pink.
6.4 Colony counting
See the colony counting part of GB 4789.2.
6.5 Result expression
See the calculation method part of GB 4789.2.
6.6 Report of colony counting
See the reporting part of total bacterial counting in GB 4789.2.
7 Result and report
Issue a report based on colony counting results. Reporting units are expressed in
CFU/g(mL).
8 Identification of lactic acid bacteria (optional)
8.1 Method one -- Biochemical identification
8.1.1 Pure culture
Pick 3 or more single colonies and inoculate streptococcus thermophilus on MC agar
plate. Place at 36°C ±1°C and incubate aerobically for 48 h. Lactobacillus are
inoculated on MRS agar plates. Place at 36°C ±1°C and incubate anaerobically for 48 h.
8.1.2 Identification of bifidobacteria
Operate according to the provisions of GB 4789.34.
8.1.3 Smear microscopy
Streptococcus thermophilus cells are spherical or club-shaped under a microscope. The
diameter is 0.5 μm~2.0 μm. They are arranged in pairs or chains, no spores, Gram stain
positive. Lactobacillus have various cell shapes under the microscope and are long rod-
shaped, curved rod-shaped or short rod-shaped, without spores, and Gram stain positive.
8.1.4 Main biochemical reactions of lactic acid bacteria
The main biochemical reactions of lactic acid bacteria strains are shown in Table 2 andTable 3.
8.2 Method two -- Real-time fluorescence PCR identification
8.2.1 Pure culture
Same as 8.1.1.
8.2.2 DNA template preparation
Use an inoculating loop to scrape 2~10 colonies on the MC agar plate or MRS agar
plate. Suspend in 200 μL of sterilized physiological saline. Mix thoroughly. Centrifuge
at 10000×g to 12000×g for 3 min. Discard the supernatant. Add 50 μL of DNA
extraction solution and vortex to mix. Place in 100℃ water bath or metal bath for 10
min and then cool quickly. Centrifuge at 10000×g ~ 12000×g for 3 min. Pipette the
supernatant into a new PCR reaction tube and use it as a DNA template. The extracted
8.2.3 PCR reaction system
The total reaction system volume is 25 μL. 2.5 μL of 10 × PCR buffer, 1 μL of upstream
and downstream primers (10 μmol/L), 0.5 μL of probe (10 μmol/L), 3 μL of dNTPs (2.5
μmol/L), Taq DNA polymerase (5 U/μL) 0.5 μL, 1 μL of template DNA. Sterilized
deionized water are added to 25 μL. Each reaction should be run in at least 2 parallels.
8.2.4 PCR reaction conditions
50°C for 5 min, 95°C for 3 min, denaturation at 94°C for 5 s, and 60°C for annealing
and extension for 40 s (collecting FAM fluorescence at the same time) for 40 cycles.
8.2.5 Control settings
During the testing process (including DNA extraction), positive controls, negative
controls, and blank controls should be set for each reaction. The positive control
template is the positive clone molecule DNA or positive strain DNA of the amplified
fragment. The negative control template is non-lactic acid bacteria strain DNA. The
blank control template is sterile water.
8.2.6 Interpretation of results
Annex A
Culture medium and reagents
A.1 Diluent
A.1.1 Ingredients
A.1.2 Preparation method
Add the above ingredients to 1000 mL of distilled water. Heat to dissolve. After
dispensing, autoclave at 121°C for 15 min.
A.2 MRS agar medium
A.2.1 Ingredients
A.2.2 Preparation method
Add the above ingredients to 1000 mL of distilled water. Heat to dissolve. Adjust the
pH to 6.2±0.2.After dispensing, autoclave at 121°C for 15 min.
A.3 Mupirocin lithium salt and cysteine hydrochloride modified MRS agar
medium
A.3.1 Preparation of mupirocin lithium salt stock solution. Weigh 50 mg of mupirocin
lithium salt and add it to 5 mL of distilled water. Sterilize by filtration with 0.22 μm
microporous filter membrane. Prepare when needed.
A.3.2 Preparation of cysteine hydrochloride stock solution. Weigh 500 mg of cysteine
hydrochloride and add it to 10 mL of distilled water. Sterilize by filtration using a 0.22
μm microporous filter membrane. Prepare when needed.
A.3.3 Preparation method
Add the ingredients in A.2.1 to 985 mL of distilled water. Heat to dissolve. Adjust the
pH to 6.2±0.2.After dispensing, autoclave at 121°C for 15 min. Heat and melt the agar
before use. Cool to 48℃~50℃ in a water bath. Use a sterile syringe to prepare the
mupirocin lithium salt stock solution and cysteine hydrochloride stock solution. Add
them to the molten agar. Make the concentration of mupirocin lithium salt in the culture
medium 50 μg/mL and the concentration of cysteine hydrochloride 500 μg/mL.
A.4 MC agar medium
A.4.1 Ingredients
A.4.2 Preparation method
Add the first 7 ingredients to the distilled water. Heat to dissolve. Adjust the pH to
6.0±0.2.Add neutral red solution. After dispensing, autoclave at 121°C for 15 min.
A.5 Lactobacillus sugar fermentation tube
A.5.1 Ingredients
......
Source: Above contents are excerpted from the full-copy PDF -- translated/reviewed by: www.ChineseStandard.net / Wayne Zheng et al.
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