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GB 4789.35-2023: National food safety standard - Food microbiological examination - Lactic acid bacteria
GB 4789.35-2023
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
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.
3.13 Sterile Erlenmeyer flask: 500 mL, 250 mL.
3.14 Sterile plate: diameter is 90 mm.
3.15 PCR tubes.
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.
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
6.2.1 Use a 1 mL sterile pipette or micropipette to draw 1 mL of the 1:10 sample
homogenate. Slowly pour along the tube wall into a sterile test tube containing 9 mL of
diluent (be careful not to touch the diluent with the tip of the pipette or micropipette
tip). Shake the test tube or use a sterile straw and pipet repeatedly to mix evenly. Make
a sample homogeneous solution of 1:100.
6.2.2 Take another 1 mL sterile pipette or micropipette tip. According to the above
operation sequence, make 10 times increments of sample homogenization. For each
incremental dilution, use a 1 mL sterile pipette or tip.
6.2.3 Foods containing lactic acid bacteria that have been treated with special
technologies (such as embedding technology) should be diluted in accordance with the
corresponding technical/process requirements.
6.3 Lactic acid bacteria counting
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.
6.3.4 Lactobacillus counting
Based on the estimate of the total number of viable bacteria 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 dilution is transferred to the plate, pour 15 mL ~ 20 mL of the MRS
agar medium cooled to 48℃~50℃ into the plate. Swirl the dish to mix evenly. After
the culture medium has solidified, place it upside down at 36°C ±1°C for anaerobic
culture. According to the growth characteristics of lactobacillus, 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 process from sample dilution to plate pouring should be completed
within 15 min.
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).
8.2 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
DNA template should be kept at 4°C for use on the same day. Otherwise, it should be
stored below -20℃ and used within 1 week.
NOTE: Depending on the actual laboratory conditions, commercial kits can also be used to prepare
DNA templates.
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.
NOTE: The amount of each reagent in the reaction system can be appropriately adjusted according
to specific conditions or different total reaction volumes. You can also choose a real-time
fluorescence PCR master mix based on Taqman probes that contains PCR buffer, MgCl2, dNTPs
and Taq enzymes.
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.
NOTE: The PCR reaction parameters can be adjusted appropriately according to the real-time
fluorescence PCR reaction system of the gene cycler model.
The primer and probe sequences for identification are shown in Table 4.